Agonistic anti-tumor necrosis factor receptor 2 antibodies

ABSTRACT

The invention provides agonistic TNFR2 antibodies and antigen-binding fragments thereof and encompasses the use of these antibodies as therapeutics to promote the proliferation of regulatory T cells (T-reg) for the treatment of immunological diseases. Antibodies of the invention can be used to potentiate the T-reg-mediated deactivation of self- and allergen-reactive T- and B-eases. Antibodies and can thus be used to treat a wide variety of indications, including autoimmune diseases, allergic reactions, asthma, graft-versus-host disease, and allograft rejection, among others.

FIELD OF THE INVENTION

The invention relates to antibodies capable of potentiating tumornecrosis factor receptor 2 signalling and their use for modulating theactivity of T-reg cells, and provides therapies for immunologicaldisorders or conditions, such as multiple sclerosis, asthma, allergicreactions, graft-versus-host disease, and transplantation graftrejection.

BACKGROUND OF THE INVENTION

Maintaining control of the cell-mediated and humoral immune responses isan important facet of healthy immune system activity. The aberrantregulation of T-cell and B-cell driven immune reactions has beenassociated with a wide array of human diseases, as the inappropriatemounting of an immune response against various self and foreign antigensplays a causal role in such pathologies as autoimmune disorders, asthma,allergic reactions, graft-versus-host disease, transplantation graftrejection, and a variety of other immunological disorders. Thesediseases are mediated by T- and B-lymphocytes that exhibit reactivityagainst self antigens and those derived from non-threatening sources,such as allergens or transplantation allografts. T-reg cells (T-regcells) have evolved in order to inhibit the activity of immune cellsthat are cross-reactive with “self” major histocompatability complex(MHC) proteins and other benign antigens. T-reg cells represent aheterogeneous class of T-cells that can be distinguished based on theirunique surface protein presentation. The most well-understoodpopulations of T-reg cells include CD4+, CD25+, FoxP3+T-reg cells andCD17+T-reg cells. The precise mechanisms by which these cells mediatesuppression of autoreactive T-cells is the subject of ongoinginvestigations, though it has been shown that certain classes of T-regcells inhibit production of the proliferation-inducing cytokine IL-2 intarget T-cells and may additionally sequester IL-2 from autoreactivecells by virtue of the affinity of CD25 (a subdomain of the IL-2receptor) for IL-2 (Josefowicz et al., Ann. Rev. Immun., 30:531-564(2012)). Moreover, it has been shown that CD4+, CD25+, FoxP3+T-reg cellsare also present in B-cell-rich areas and are capable of directlysuppressing immunoglobulin production independent of their ability toattenuate TH2-cell activity (Lim et al., J. Immunol., 175:4180-4183(2005)).

Tumor necrosis factor receptor (TN FR) subtypes 1 and 2 have beenidentified on the T-reg cell surface as signal transduction moleculesthat dictate cell fate. The activation of TNFR1, for instance,potentiates the caspase signaling cascade and terminates in T-regapoptosis, while activation of TNFR2 induces signaling through themitogen-activated protein kinase (MAPK) signaling pathway, whichorchestrates the TRAF2/3- and NFκB-mediated transcription of genes thatpromote cell proliferation and escape from apoptosis. Due to its role indirecting cell survival and growth, TNFR2 represents an attractivetarget for expanding populations of T-reg cells as a strategy fortreating immunological disorders. There is currently a need fortherapies that can augment T-reg cell survival and proliferation for usein treatments targeting such diseases as autoimmune disorders,graft-versus-host disease, allograft rejection, allergic reactions, andasthma, among others.

SUMMARY OF THE INVENTION

The invention provides TNFR2 agonist antibodies and antigen-bindingfragments thereof capable of binding TNFR2 and promoting TNFR2signaling, as well as methods of producing such antibodies andantigen-binding fragments thereof, and methods of treating a subjectsuffering from an immunological disease by administering such antibodiesand antigen-binding fragments thereof.

In a first aspect, the invention provides a TNFR2 agonist antibody orantigen-binding fragment thereof capable of specifically binding TNFR2(e.g., human TNFR2), e.g., in a human or in a non-human animal. Theantibody or antigen-binding fragment thereof specifically binds anepitope containing amino acids 56-60 of SEQ ID NO: 366 (KCSPG) and doesnot specifically bind an epitope containing amino acids 142-146 of SEQID NO: 366 (KCRPG). The antibody or antigen-binding fragment thereof mayalso lack specific binding for another tumor necrosis factor receptor(TNFR) superfamily member, such as TNFR1, RANK, CD30, CD40, Lymphotoxinbeta receptor (LT-PR), OX40, Fas receptor, Decoy receptor 3, CD27, 4-1BB, Death receptor 4, Death receptor 5, Decoy receptor 1, Decoy receptor2, Osteoprotegrin, TWEAK receptor, TACI, BAFF receptor, Herpesvirusentry mediator, Nerve growth factor receptor, B-cell maturation antigen,Glucocorticoid-induced TNFR-related, TROY, Death receptor 6, Deathreceptor 3, and Ectodysplasin A2 receptor. TNFR2 agonist antibodies andantigen-binding fragments thereof that specifically bind non-human TNFR2exhibit specific binding to an epitope containing amino acids KCPPG, butdo not specifically bind an epitope containing amino acids KCGPG and/orKCSPG. In addition, TNFR2 agonist antibodies and antigen-bindingfragments thereof that specifically bind non-human TNFR2 may also lackspecific binding to a TNFR superfamily member other than TNFR2.

In an additional aspect, the invention provides a TNFR2 agonist antibodyor antigen-binding fragment thereof capable of specifically bindingTNFR2, such as human TNFR2, that specifically binds an epitopecontaining amino acids 56-60 of SEQ ID NO: 366 (KCSPG) and does notspecifically bind another TNFR superfamily member. In certainembodiments, the antibody or antigen-binding fragment thereof does notbind an epitope containing amino acids 142-146 of SEQ ID NO: 366(KCRPG). In additional cases, the antibody or antigen-binding fragmentthereof does not specifically bind any other epitope within TNFR2.

The invention also provides a TNFR2 agonist antibody or antigen-bindingfragment thereof capable of specifically binding TNFR2, such as humanTNFR2, that specifically binds an epitope containing amino acids 56-60of SEQ ID NO: 366 (KCSPG) and is capable of promoting the proliferationof a population of T-regulatory (T-reg) cells. In another aspect, theinvention encompasses a TNFR2 agonist antibody or antigen-bindingfragment thereof capable of specifically binding TNFR2, such as humanTNFR2, that specifically binds an epitope containing amino acids 56-60of SEQ ID NO: 366 (KCSPG) and is capable of promoting the death of oneor more CD8+ T-cells. In yet another aspect, the invention provides aTNFR2 agonist antibody or antigen-binding fragment thereof capable ofspecifically binding TNFR2, such as human TNFR2, that specifically bindsan epitope containing amino acids 56-60 of SEQ ID NO: 366 (KCSPG) and iscapable of promoting an increase in the level of one or more mRNAmolecules encoding a protein selected from the group consisting ofcIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, a protein involved in theangiogenic pathway, an IKK complex, RIP, NIK, MAP3K, a protein involvedin the NFkB pathway, NIK, JNK, AP-1, a MEK (e.g., MEK1, MEK7), MKK3,NEMO, IL2R, Foxp3, IL2, TNF, and lymphotoxin (e.g., lymphotoxin α andlymphotoxin β). In another aspect, the invention provides a TNFR2agonist antibody or antigen-binding fragment thereof capable ofspecifically binding TNFR2, such as human TNFR2, that specifically bindsan epitope amino acids 56-60 of SEQ ID NO: 366 (KCSPG) and is capable ofpromoting an increase in the level of one or more proteins selected fromthe group consisting of cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, a proteininvolved in the angiogenic pathway, an IKK complex, RIP, NIK, MAP3K, aprotein involved in the NFkB pathway, NIK, JNK, AP-1, a MEK (e.g., MEK1,MEK7), MKK3, NEMO, IL2R, Foxp3, IL2, TNF, and lymphotoxin (e.g.,lymphotoxin α and lymphotoxin β).

The TNFR2 agonist antibody or antigen-binding fragment thereof of theinvention specifically binds an epitope within human TNFR2 containing atleast five discontinuous or continuous residues within amino acids96-154 of SEQ ID NO: 366(CGSRCSSDQVETQACTREONRICTCRPGWYCALSKQESCRLCAPLRKCRPGFGVARPGT).Additionally or alternatively, the antibody or antigen-binding fragmentthereof binds an epitope within amino acids 111-150 of SEQ ID NO: 366(TREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA). In other cases, the antibodyor antigen-binding fragment thereof binds an epitope within amino acids115-142 of SEQ ID NO: 366 (NRICTCRPGWYCALSKQEGCRLCAPLRK). Antibodies andantigen-binding fragments of the invention may also bind an epitopewithin amino acids 122-136 of SEQ ID NO: 366 (PGWYCALSKQEGCRL), and/oran epitope within amino acids 101-107 of SEQ ID NO: 366 (SSDQVET). Infurther embodiments, the antibody or antigen-binding fragment thereof ofthe invention binds an epitope within amino acids 48-67 of SEQ ID NO:366 (QTAQMCCSKCSPGQHAKVFC). In some cases, the antibody orantigen-binding fragment thereof of the invention specifically binds theepitope containing amino acids 56-60 of SEQ ID NO: 366 (KCSPG) with aK_(D) of less than about 10 nM.

In another aspect, the invention provides a TNFR2 agonist antibody orantigen-binding fragment thereof that specifically binds to an epitopewithin or containing the amino acid sequence of any one of SEQ ID NOs:1-341, 346, and 367 and that is capable of specifically binding humanTNFR2 but does not specifically bind another TNFR superfamily member.

The TNFR2 agonist antibody or antigen-binding fragment thereof mayactivate TNFR2 signaling. The antibody or antigen-binding fragmentthereof may also bind TNFR2 with a K_(D) of no greater than about 10 nM(e.g., with a K_(D) of no greater than about 1 nM). Additionally oralternatively, the antibody or antigen-binding fragment thereof bindsTNFR2 to form an antibody-antigen complex with a k_(on) of at leastabout 10⁴ M⁻¹s⁻¹ (e.g., with a k_(on) of at least about 10⁵ M⁻¹s⁻¹). Insome cases, the antibody or antigen-binding fragment thereof binds TNFR2to form an antibody-antigen complex, in which the complex dissociateswith a k_(off) of no greater than about 10⁻³ s⁻¹ (e.g., with a k_(off)of no greater than about 10⁻⁴ s⁻¹). In additional embodiments, theantibody or antigen-binding fragment thereof is capable of promoting theproliferation of a population of T-regulatory (T-reg) cells (e.g., inthe presence of TNFα).

In some embodiments, the antibody or antigen-binding fragment thereofhas a non-native constant region. For instance, the antibody may be amonoclonal antibody that has a non-native constant region. In someembodiments, the antibody or antigen-binding fragment thereof is anisolated, non-murine antibody.

In another aspect, the invention provides a method of identifying aTNFR2 agonist antibody or antigen-binding fragment thereof, the methodincluding the steps of:

(a) contacting a mixture of antibodies or fragments thereof with atleast one peptide having the amino acid sequence of any one of SEQ IDNOs: 1-341, 346, and 367; and

(b) separating antibodies or fragments thereof that specifically bindthe peptide from the mixture, thereby producing an enriched antibodymixture comprising at least one TNFR2 agonist antibody orantigen-binding fragment thereof.

In some cases, the method includes the step of determining the aminoacid sequence of one or more of the antibodies or antigen-bindingfragments thereof in the enriched antibody mixture. In certainembodiments, the peptide is bound to a surface. Additionally oralternatively, the antibody or antigen-binding fragment thereof isexpressed on the surface of a phage, bacterial cell, or yeast cell. Inother cases, the antibody or antigen-binding fragment thereof isexpressed as one or more polypeptide chains non-covalently bound toribosomes or covalently bound to mRNA or cDNA. The peptide may beconjugated to a detectable label, such as a fluorescent molecule (e.g.,green fluorescent protein, cyan fluorescent protein, yellow fluorescentprotein, red fluorescent protein, phycoerythrin, allophycocyanin,hoescht, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide,fluorescein, coumarin, rhodamine, tetramethylrhoadmine, and cyanine) oran epitope tag (e.g., maltose-binding protein,glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag,human influenza hemagglutinin (HA) tag, biotin, and streptavidin). Insome embodiments, steps (a) and (b) described above are sequentiallyrepeated one or more times.

In additional embodiments of the invention, the method further includesthe steps of:

i) exposing the enriched antibody mixture to at least one peptidecontaining the amino acid sequence of a TNFR superfamily member otherthan TNFR2; and retaining antibodies or fragments thereof that do notspecifically bind the peptide, thereby producing a TNFR2-specificantibody mixture containing at least one TNFR2 agonist antibody orantigen-binding fragment thereof that does not specifically bind a TNFRsuperfamily member other than TNFR2; and/or

ii) exposing the enriched antibody mixture to at least one peptidecontaining amino acids 142-146 of SEQ ID NO: 366 (KCRPG); and retainingantibodies or fragments thereof that do not specifically bind thepeptide, thereby producing an antibody mixture containing at least oneTNFR2 agonist antibody or antigen-binding fragment thereof that does notspecifically bind a peptide containing amino acids 142-146 of SEQ ID NO:366 (KCRPG).

In some cases, the method includes performing steps (i) and (ii) ineither order.

In another aspect, the invention provides a method of producing a TNFR2agonist antibody or antigen-binding fragment thereof by immunizing anon-human mammal with a peptide containing the sequence of any one ofSEQ ID NOs: 1-341, 346, and 367 and collecting serum containing theTNFR2 agonist antibody or antigen-binding fragment thereof, such thatthe antibody or antigen-binding fragment thereof is capable ofspecifically binding an epitope containing amino acids 56-60 of SEQ IDNO: 366 (KCSPG). In some cases, the non-human mammal is selected fromthe group consisting of a rabbit, mouse, rat, goat, guinea pig, hamster,horse, primate, and sheep. Additionally or alternatively, the peptidecontains the amino acid sequence PGWYCALSKQEGCRL (SEQ ID NO: 11).

In a further aspect, the invention provides a TNFR2 agonist antibody orantigen-binding fragment thereof produced by any of the above-describedmethods. In some cases, the antibody or antigen-binding fragment thereofspecifically binds an epitope containing amino acids 56-60 of SEQ ID NO:366 (KCSPG) and does not specifically bind an epitope containing aminoacids 142-146 of SEQ ID NO: 366 (KCRPG). Additionally or alternatively,the antibody or antigen-binding fragment thereof specifically binds anepitope containing amino acids 56-60 of SEQ ID NO: 366 (KCSPG) and doesnot specifically bind a TNFR superfamily member other than TNFR2. Insome cases, the antibody or antigen-binding fragment thereof activatesTNFR2 signaling. In additional embodiments, the antibody orantigen-binding fragment thereof binds TNFR2 with a K_(D) of no greaterthan about 10 nM (e.g., with a K_(D) of no greater than about 1 nM).Additionally or alternatively, the antibody or antigen-binding fragmentthereof binds TNFR2 to form an antibody-antigen complex with a k_(on) ofat least about 10⁴ M⁻¹s⁻¹ (e.g., with a k_(on) of at least about 10⁵M⁻¹s⁻¹). In some cases, the antibody or antigen-binding fragment thereofbinds TNFR2 to form an antibody-antigen complex, in which the complexdissociates with a k_(off) of no greater than about 10⁻³s⁻¹ (e.g., witha k_(off) of no greater than about 10⁻⁴s⁻¹). In additional embodiments,the antibody or antigen-binding fragment thereof is capable of promotingthe proliferation of a population of T-regulatory (T-reg) cells (e.g.,in the presence of TNFα).

In some cases, the TNFR2 agonist antibody or antigen-binding fragmentthereof of the invention is a monoclonal antibody or antigen-bindingfragment thereof, a polyclonal antibody or antigen-binding fragmentthereof, a human antibody or antigen-binding fragment thereof, ahumanized antibody or antigen-binding fragment thereof, a primatizedantibody or antigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, or a multi-specific antibody orantigen-binding fragment thereof. In other embodiments, the antibody orantigen-binding fragment thereof of the invention is a dual-variableimmunoglobulin domain, a monovalent antibody or antigen-binding fragmentthereof, a chimeric antibody or antigen-binding fragment thereof, asingle-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a domain antibody, a Fv fragment, a Fabfragment, a F(ab′)₂ molecule, or a tandem scFv (taFv).

In further embodiments, the TNFR2 agonist antibody of the invention maycontain an immunoglobulin, such as an immunoglobulin of subtype IgG,IgM, IgA, IgD, or IgE.

In some embodiments, the antibody or antigen-binding fragment thereofhas a non-native constant region. For instance, the antibody may be amonoclonal antibody that has a non-native constant region. In someembodiments, the antibody or antigen-binding fragment thereof is anisolated, non-murine antibody.

In another aspect, the invention provides a pharmaceutical compositioncontaining a TNFR2 agonist antibody or antigen-binding fragment thereofof the invention and a pharmaceutically acceptable carrier or excipient.The pharmaceutical composition of the invention may also contain anadditional therapeutic agent, such as TNFα or BCG, or an immunotherapyagent, such as an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1agent, an anti-PD-L2 agent, a TNF-α cross-linking agent, a TRAILcross-linking agent, a CD27 agent, a CD30 agent, a CD40 agent, a 4-1 BBagent, a GITR agent, an OX40 agent, a TRAILR1 agent, a TRAILR2 agent, ora TWEAKR agent.

The invention also provides a polynucleotide encoding a TNFR2 agonistantibody or antigen-binding fragment thereof of the invention, as wellas a vector containing such a polynucleotide. In some embodiments, thevector is an expression vector, such as a eukaryotic expression vector.In other embodiments, the expression vector is a viral vector, such asan adenovirus (Ad, e.g., a serotype 5, 26, 35, or 48 adenovirus),retrovirus (e.g., a γ-retrovirus or a lentivirus), poxvirus,adeno-associated virus, baculovirus, herpes simplex virus, or a vacciniavirus (e.g., a modified vaccinia Ankara (MVA)).

In a further aspect, the invention encompasses a host cell containing avector of the invention. In some cases, the host cell is a prokaryoticcell. In other embodiments, the vector is a eukaryotic cell, such as amammalian cell (e.g., a CHO cell, a DHFR CHO cell, a NSO myeloma cell, aCOS cell, a 293 cell, or a SP2/0 cell.).

The invention additionally provides a method of producing a TNFR2agonist antibody or antigen-binding fragment described above, the methodincluding the steps of expressing a polynucleotide encoding the antibodyor antigen-binding fragment thereof in a host cell and recovering theantibody or antigen-binding fragment thereof from host cell medium.

In another aspect, the invention provides a method of inhibiting animmune response mediated by a B cell or a CD8+ T cell in a subject and amethod of treating an immunological disease in a subject, the methodsindividually including the step of administering to the subject a TNFR2agonist antibody or antigen binding fragment of the invention, apharmaceutical composition of the invention, a polynucleotide of theinvention, a vector of the invention, or a host cell of the invention.

In some embodiments, the antibody or antigen-binding fragment thereofhas a non-native constant region. For instance, the antibody may be amonoclonal antibody that has a non-native constant region. In someembodiments, the antibody or antigen-binding fragment thereof is anisolated, non-murine antibody.

In some cases, the subject is in need of tissue or organ repair orregeneration (e.g., repair or regeneration of a pancreas, salivarygland, pituitary gland, kidney, heart, lung, hematopoietic system,cranial nerves, heart, aorta, olfactory gland, ear, nerves, structuresof the head, eye, thymus, tongue, bone, liver, small intestine, largeintestine, gut, lung, brain, skin, peripheral nervous system, centralnervous system, spinal cord, breast, embryonic structures, embryos, andtestes). Administration of a TNFR2 agonist antibody or antigen-bindingfragment thereof stimulates or allows repair and/or regeneration of thetissue or organ.

The immunological disease to be treated may be selected from the groupconsisting of an autoimmune disease, a neurological condition, anallergy, asthma, macular degeneration, muscular atrophy, a diseaserelated to miscarriage, atherosclerosis, bone loss, a musculoskeletaldisease, obesity, a graft-versus-host disease, and an allograftrejection. In other embodiments, the autoimmune disease is selected fromthe group consisting of type I diabetes, Alopecia Areata, AnkylosingSpondylitis, Antiphospholipid Syndrome, Autoimmune Addison's Disease,Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Behcet's Disease,Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, ChronicFatigue Immune Dysfunction Syndrome (CFIDS), Chronic InflammatoryDemyelinating Polyneuropathy, Churg-Strauss Syndrome, CicatricialPemphigoid, CREST Syndrome, Cold Agglutinin Disease, Crohn's Disease,Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves'Disease, Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism,Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura(ITP), IgA Nephropathy, Juvenile Arthritis, Lichen Planus, Lupus,Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis,Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, PolyarteritisNodosa, Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, PrimaryBiliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma,Sjögren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis,Vitiligo, and Wegener's Granulomatosis.

In other embodiments, the neurological condition is selected from thegroup consisting of a brain tumor, a brain metastasis, a spinal cordinjury, schizophrenia, epilepsy, Amyotrophic lateral sclerosis (ALS),Parkinson's disease, Alzheimer's disease, Huntington's disease, andstroke.

In some cases, the allergy is selected from the group consisting of foodallergy, seasonal allergy, pet allergy, hives, hay fever, allergicconjunctivitis, poison ivy allergy oak allergy, mold allergy, drugallergy, dust allergy, cosmetic allergy, and chemical allergy.

In other embodiments, the allograft rejection is selected from the groupconsisting of skin graft rejection, bone graft rejection, vasculartissue graft rejection, ligament graft rejection (e.g., cricothyroidligament graft rejection, periodontal ligament graft rejection,suspensory ligament of the lens graft rejection, palmar radiocarpalligament graft rejection, dorsal radiocarpal ligament graft rejection,ulnar collateral ligament graft rejection, radial collateral ligamentgraft rejection, suspensory ligament of the breast graft rejection,anterior sacroiliac ligament graft rejection, posterior sacroiliacligament graft rejection, sacrotuberous ligament graft rejection,sacrospinous ligament graft rejection, inferior pubic ligament graftrejection, superior pubic ligament graft rejection, anterior cruciateligament graft rejection, lateral collateral ligament graft rejection,posterior cruciate ligament graft rejection, medial collateral ligamentgraft rejection, cranial cruciate ligament graft rejection, caudalcruciate ligament graft rejection, and patellar ligament graftrejection), and organ graft rejection.

In still other embodiments, the graft-versus-host disease arises from abone marrow transplant or one or more blood cells selected from thegroup consisting of hematopoietic stem cells, common myeloid progenitorcells, common lymphoid progenitor cells, megakaryocytes, monocytes,basophils, eosinophils, neutrophils, macrophages, T-cells, B-cells,natural killer cells, and dendritic cells.

In some cases, the above-described methods include administering to thesubject an additional therapeutic agent, such as TNFα or BCG.Additionally or alternatively, the subject may be administered animmunotherapy agent, such as an anti-CTLA-4 agent, an anti-PD-1 agent,an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α cross-linking agent, aTRAIL cross-linking agent, a CD27 agent, a CD30 agent, a CD40 agent, a4-1 BB agent, a GITR agent, an OX40 agent, a TRAILR1 agent, a TRAILR2agent, or a TWEAKR agent.

In certain embodiments of the above-described methods, the subject is amammal (e.g., a human). Additionally or alternatively, the antibody maybe 8E6.D1 or a humanized antibody or antigen-binding fragment thereofcontaining one or more (or all) heavy chain and/or light chain CDRs of8E6.D1.

The invention also provides a kit containing an agent, such as a TNFR2agonist antibody or antigen binding fragment of the invention, apharmaceutical composition of the invention, a polynucleotide of theinvention, a vector of the invention, and/or a host cell of theinvention. In some cases, the kit includes instructions for transfectinga vector of the invention into a host cell. Additionally, the kit mayinclude instructions for expressing a TNFR2 agonist antibody orantigen-binding fragment thereof of the invention in the host cell,and/or a reagent that can be used to express the antibody orantigen-binding fragment thereof in the host cell. In other embodiments,the kit includes instructions for administering the agent to a subject(e.g., a mammalian subject, such as a human) in order to treat animmunological disease. In other embodiments, the kit includesinstructions for making or using the agent.

Definitions

As used herein, the term “about” refers to a value that is no more than10% above or below the value being described. For example, the term“about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.

As used herein, the terms “agonist TNFR2 antibody” and “agonistic TNFR2antibody” refer to TNFR2 antibodies that are capable of promoting orincreasing activation of TNFR2 and/or potentiating one or more signaltransduction pathways mediated by TNFR2. For example, agonistic TNFR2antibodies of the invention can promote or increase the proliferation ofa population of T-reg cells. Agonistic TNFR2 antibodies of the inventionmay promote or increase TNFR2 activation by binding TNFR2, e.g., so asto induce a conformational change that renders the receptor biologicallyactive. For instance, agonistic TNFR2 antibodies may nucleate thetrimerization of TNFR2 in a manner similar to the interaction betweenTNFR2 and its cognate ligand, TNFα, thus inducing TNFR2-mediatedsignalling. Agonistic TNFR2 antibodies of the invention may be capableof inducing the proliferation of CD4+, CD25+, FOXP3+T-reg cells.Agonistic TNFR2 antibodies of the invention may also be capable ofsuppressing the proliferation of cytotoxic T lymphocytes (e.g., CD8+T-cells), e.g., through activation of immunomodulatory T-reg cells or bydirectly binding TNFR2 on the surface of an autoreactive cytotoxicT-cell and inducing apoptosis. Unless otherwise noted, the terms“agonist TNFR2 antibody” and “agonistic TNFR2 antibody” also includeantibody fragments, e.g., those described below, that retain the abilityto bind TNFR2 and potentiate TNFR2 signal transduction. An agonist TNFR2antibody or fragment thereof may specifically bind TNFR2 withoutexhibiting specific binding for another receptor of the tumor necrosisfactor receptor (TNFR) superfamily.

As used herein, the term “antibody” (Ab) refers to an immunoglobulinmolecule that specifically binds to, or is immunologically reactivewith, a particular antigen, and includes polyclonal, monoclonal,genetically engineered and otherwise modified forms of antibodies,including but not limited to chimeric antibodies, humanized antibodies,heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies,diabodies, triabodies, and tetrabodies), and antigen-binding fragmentsof antibodies, including e.g., Fab′, F(ab′)₂, Fab, Fv, recombinant IgG(rIgG) fragments, and scFv fragments. Moreover, unless otherwiseindicated, the term “monoclonal antibody” (mAb) is meant to include bothintact molecules, as well as antibody fragments (such as, for example,Fab and F(ab′)₂ fragments) that are capable of specifically binding to atarget protein. Fab and F(ab′)₂ fragments lack the Fc fragment of anintact antibody, clear more rapidly from the circulation of the animal,and may have less non-specific tissue binding than an intact antibody(see Wahl et al., J. Nucl. Med. 24:316, 1983; incorporated herein byreference).

The term “antigen-binding fragment,” as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to a target antigen. The antigen-binding function of an antibodycan be performed by fragments of a full-length antibody. The antibodyfragments can be, e.g., a Fab, F(ab′)₂, scFv, SMIP, diabody, a triabody,an affibody, a nanobody, an aptamer, or a domain antibody. Examples ofbinding fragments encompassed by the term “antigen-binding fragment” ofan antibody include, but are not limited to: (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L), and C_(H)1domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb including V_(H) and V_(L) domains; (vi) a dAbfragment (Ward et al., Nature 341:544-546, 1989), which consists of aV_(H) domain; (vii) a dAb which consists of a V_(H) or a V_(L) domain;(viii) an isolated complementarity determining region (CDR); and (ix) acombination of two or more isolated CDRs which may optionally be joinedby a synthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv (scFv); see,e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments canbe obtained using conventional techniques known to those of skill in theart, and the fragments can be screened for utility in the same manner asintact antibodies. Antigen-binding fragments can be produced byrecombinant DNA techniques, enzymatic or chemical cleavage of intactimmunoglobulins, or, in certain cases, by chemical peptide synthesisprocedures known in the art.

As used herein, the terms “anti-tumor necrosis factor receptor 2antibody,” “TNFR2 antibody,” “anti-TNFR2 antibody portion,” and/or“anti-TNFR2 antibody fragment” and the like include any protein orpeptide-containing molecule that includes at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that is capable of specifically bindingto TNFR2. TNFR2 antibodies also include antibody-like protein scaffolds,such as the tenth fibronectin type III domain (¹⁰Fn3), which containsBC, DE, and FG structural loops similar in structure and solventaccessibility to antibody CDRs. The tertiary structure of the ¹⁰Fn3domain resembles that of the variable region of the IgG heavy chain, andone of skill in the art can graft, e.g., the CDRs of a TNFR2 monoclonalantibody onto the fibronectin scaffold by replacing residues of the BC,DE, and FG loops of ¹⁰Fn3 with residues from the CDRH-1, CDRH-2, orCDRH-3 regions of a TNFR2 monoclonal antibody. The use of ¹⁰Fn3 domainsas scaffolds for epitope grafting is described, e.g., in WO 2000/034784,the disclosure of which is incorporated herein by reference. Additionalscaffold proteins encompassed by the term “anti-tumor necrosis factorreceptor 2 antibody,” “TNFR2 antibody,” and the like include peptide-Fcfusion proteins (described, e.g., in WO 2012/122378; as well as in U.S.Pat. No. 8,633,297; the disclosures of each of which are incorporatedherein by reference).

As used herein, the term “bispecific antibodies” refers to monoclonal,often human or humanized antibodies that have binding specificities forat least two different antigens. Bispecific TNFR2 antibodies of theinvention may have binding specificities that are directed towards TNFR2and any other antigen, e.g., for a cell-surface protein, receptor,receptor subunit, tissue-specific antigen, virally derived protein,virally encoded envelope protein, bacterially derived protein, orbacterial surface protein, etc. A bispecific antibody may also be anantibody or antigen-binding fragment thereof that includes two separateantigen-binding domains (e.g., two scFvs joined by a linker). The scFvsmay bind the same antigen or different antigens.

As used herein, the term “chimeric” antibody refers to an antibodyhaving variable sequences derived from an immunoglobulin of one sourceorganism, such as rat or mouse, and constant regions derived from animmunoglobulin of a different organism (e.g., a human). Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229(4719): 1202-7; Oi et al, 1986, BioTechniques4:214-221; Gillies et al, 1985, J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397; the disclosures of eachof which are incorporated herein by reference.

As used herein, the term “complementarity determining region” (CDR)refers to a hypervariable region found both in the light chain and theheavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). As isappreciated in the art, the amino acid positions that delineate ahypervariable region of an antibody can vary, depending on the contextand the various definitions known in the art. Some positions within avariable domain may be viewed as hybrid hypervariable positions in thatthese positions can be deemed to be within a hypervariable region underone set of criteria while being deemed to be outside a hypervariableregion under a different set of criteria. One or more of these positionscan also be found in extended hypervariable regions. The inventionincludes antibodies comprising modifications in these hybridhypervariable positions. The variable domains of native heavy and lightchains each comprise four framework regions that primarily adopt aβ-sheet configuration, connected by three CDRs, which form loops thatconnect, and in some cases form part of, the β-sheet structure. The CDRsin each chain are held together in close proximity by the FR regions inthe order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from theother antibody chains, contribute to the formation of the target bindingsite of antibodies (see Kabat et al, Sequences of Proteins ofImmunological Interest (National Institute of Health, Bethesda, Md.1987; incorporated herein by reference). As used herein, numbering ofimmunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat et al,unless otherwise indicated.

As used herein, the terms “conservative mutation,” “conservativesubstitution,” or “conservative amino acid substitution” refer to asubstitution of one or more amino acids for one or more different aminoacids that exhibit similar physicochemical properties, such as polarity,electrostatic charge, and/or steric volume. These properties aresummarized for each of the twenty naturally-occurring amino acids inTable 1 below.

TABLE 1 Representative physicochemical properties of naturally-occurringamino acids Charge of side Side- chain at 3 Letter 1 Letter chain pH ofSteric Amino Acid Code Code Polarity 7.4 Volume^(†) Alanine Ala Anonpolar neutral small Arginine Arg R polar positive large AsparagineAsn N polar neutral intermediate Aspartic acid Asp D polar negativeintermediate Cysteine Cys C nonpolar neutral intermediate Glutamic acidGlu E polar negative intermediate Glutamine Gln Q polar neutralintermediate Glycine Gly G nonpolar neutral small Histidine His H polarneutral large (90%) Isoleucine Ile I nonpolar neutral large Leucine LeuL nonpolar neutral large Lysine Lys K polar positive large MethionineMet M nonpolar neutral large Phenylalanine Phe F nonpolar neutral largeProline Pro P nonpolar neutral intermediate Serine Ser S polar neutralsmall Threonine Thr T polar neutral intermediate Tryptophan Trp Wnonpolar neutral bulky Tyrosine Tyr Y polar neutral large Valine Val Vnonpolar neutral intermediate ^(†)based on volume in A³: 50-100 issmall, 100-150 is intermediate, 150-200 is large, and >200 is bulky

From this table it is appreciated that the conservative amino acidfamilies include (i) G, A, V, L and I; (ii) D and E; (iii) A, S and T;(iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservativemutation or substitution is therefore one that substitutes one aminoacid for a member of the same amino acid family (e.g., a substitution ofSer for Thr or Lys for Arg).

As used herein, the term “conjugate” refers to a compound formed by thechemical bonding of a reactive functional group of one molecule with anappropriately reactive functional group of another molecule. Conjugatesmay additionally be produced, e.g., as two polypeptide domainscovalently bound to one another as part of a single polypeptide chainthat is synthesized by the translation of a single RNA transcriptencoding both polypeptides in frame with one another.

As used herein, the term “derivatized antibodies” refers to antibodiesthat are modified by a chemical reaction so as to cleave residues or addchemical moieties not native to an isolated antibody. Derivatizedantibodies can be obtained, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by addition ofknown chemical protecting/blocking groups, proteolytic cleavage, linkageto a cellular ligand or other protein. Any of a variety of chemicalmodifications can be carried out by known techniques, including, withoutlimitation, specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. using established procedures.Additionally, the derivative can contain one or more non-natural aminoacids, e.g., using amber suppression technology (see, e.g., U.S. Pat.No. 6,964,859; incorporated herein by reference). In certain cases, itmay be desirable to include one or more non-natural amino acids withinan antibody of the invention in order to provide a reactive functionalgroup that can be used to conjugate the antibody to another molecule.Examples of unnatural amino acids that exhibit this functionalityinclude those that contain, e.g., one or more azide, alkyne, ketone,aniline, alkene, tetrazole, 1,2-aminothiol, phosphine, norbornene, ortetrazine moieties. The reactivity of these functional groups is knownto those of skill in the art and is described, e.g., in US 2015/0005481;U.S. Pat. No. 7,807,619; de Araújo, et al., Chemistry 12:6095-6109(2006); and Köhn, et al., Angew Chem Int Ed Engl, 43:3106-3116 (2004);the disclosures of each of which are incorporated herein by reference.Other examples of non-natural amino acids that may desirably beincorporated into an antibody of the invention include D-amino acids andother those containing other non-natural side-chain moieties so as toreduce the susceptibility of the antibody to proteolytic degradation byevading recognition by endogenous proteases and endopeptidases.

As used herein, the term “diabodies” refers to bivalent antibodiescomprising two polypeptide chains, in which each polypeptide chainincludes V_(H) and V_(L) domains joined by a linker that is too short(e.g., a linker composed of five amino acids) to allow forintramolecular association of V_(H) and V_(L) domains on the samepeptide chain. This configuration forces each domain to pair with acomplementary domain on another polypeptide chain so as to form ahomodimeric structure. Accordingly, the term “triabodies” refers totrivalent antibodies comprising three peptide chains, each of whichcontains one V_(H) domain and one V_(L) domain joined by a linker thatis exceedingly short (e.g., a linker composed of 1-2 amino acids) topermit intramolecular association of V_(H) and V_(L) domains within thesame peptide chain. In order to fold into their native structure,peptides configured in this way typically trimerize so as to positionthe V_(H) and V_(L) domains of neighboring peptide chains spatiallyproximal to one another to permit proper folding (see Holliger et al.,Proc. Natl. Acad. Sci. USA 90:6444-48, 1993; incorporated herein byreference).

As used herein, a “dual variable domain immunoglobulin” (“DVD-Ig”)refers to an antibody that combines the target-binding variable domainsof two monoclonal antibodies via linkers to create a tetravalent,dual-targeting single agent. (Gu et al., Meth. Enzymol., 502:25-41,2012; incorporated by reference herein). Suitable linkers for use in thelight chains of the DVDs of the invention include those identified onTable 2.1 on page 30 of Gu et al.: the short K chain linkers ADAAP (SEQID NO: 380) (murine) and TVAAP (SEQ ID NO: 381) (human); the long Kchain linkers ADAAPTVSIFP (SEQ ID NO: 382) (murine) and TVAAPSVFIFPP(SEQ ID NO: 383) (human); the short A chain linker QPKAAP (SEQ ID NO:384 (human); the long A chain linker QPKAAPSVTLFPP (SEQ ID NO: 385)(human); the GS-short linker GGSGG (SEQ ID NO: 386), the GS-mediumlinker GGSGGGGSG (SEQ ID NO: 387), and the GS-long linker GGSGGGGSGGGGS(SEQ ID NO: 388) (all GS linkers are murine and human). Suitable linkersfor use in the heavy chains of the DVDs include those identified onTable 2.1 on page 30 of Gu & Ghayur, 2012, Methods in Enzymology502:25-41, incorporated by reference herein: the short linkers AKTTAP(SEQ ID NO: 389) (murine) and ASTKGP (SEQ ID NO: 390) (human); the longlinkers AKTTAPSVYPLAP (SEQ ID NO: 391) (murine) and ASTKGPSVFPLAP (SEQID NO: 392) (human); the GS-short linker GGGGSG (SEQ ID NO: 393), theGS-medium linker GGGGSGGGGS (SEQ ID NO: 394), and the GS-long linkerGGGGSGGGGSGGGG (SEQ ID NO: 395) (all GS linkers are murine and human).

As used herein, the term “endogenous” describes a molecule (e.g., apolypeptide, protein, antibody, enzyme, cofactor, or nucleic acid) thatis found naturally in a particular organism (e.g., a human) or in aparticular location within an organism (e.g., an organ, a tissue, or acell, such as a human cell).

As used herein, the term “exogenous” describes a molecule (e.g., apolypeptide, protein, antibody, enzyme, cofactor, or nucleic acid) thatis not found naturally in a particular organism (e.g., a human) or in aparticular location within an organism (e.g., an organ, a tissue, or acell, such as a human cell). Exogenous materials include those that areprovided from an external source to an organism or to cultured matterextracted there from.

As used herein, the term “framework region” or “FR” includes amino acidresidues that are adjacent to the CDRs. FR residues may be present in,for example, human antibodies, rodent-derived antibodies (e.g., murineantibodies), humanized antibodies, primatized antibodies, chimericantibodies, antibody fragments (e.g., Fab fragments), single chainantibody fragments (e.g., scFv fragments), antibody domains, andbispecific antibodies, among others.

As used herein, the term “fusion protein” refers to a protein that isjoined via a covalent bond to another molecule. A fusion protein can bechemically synthesized by, e.g., an amide-bond forming reaction betweenthe N-terminus of one protein to the C-terminus of another protein.Alternatively, a fusion protein containing one protein or protein domaincovalently bound to another protein or protein domain can be expressedrecombinantly in a cell (e.g., a eukaryotic cell or prokaryotic cell) byexpression of a polynucleotide encoding the fusion protein, for example,from a vector or the genome of the cell. A fusion protein may containone protein that is covalently bound to a linker, which in turn iscovalently bound to another molecule. Examples of linkers that can beused for the formation of a fusion protein include peptide-containinglinkers, such as those that contain naturally occurring or non-naturallyoccurring amino acids. In certain cases, it may be desirable to includeD-amino acids in the linker, as these residues are not present innaturally-occurring proteins and are thus more resistant to degradationby endogenous proteases. Linkers can be prepared using a variety ofstrategies that are well known in the art, and depending on the reactivecomponents of the linker, can be cleaved by enzymatic hydrolysis,photolysis, hydrolysis under acidic conditions, hydrolysis under basicconditions, oxidation, disulfide reduction, nucleophilic cleavage, ororganometallic cleavage. These and other linker modalities aredescribed, e.g., in Leriche et al., Bioorg. Med. Chem., 20:571-582,(2012), the disclosure of which is incorporated herein by reference.

As used herein, the term “heterospecific antibodies” refers tomonoclonal, preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. Traditionally, therecombinant production of heterospecific antibodies is based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different specificities (as described, e.g.,in Milstein et al., Nature 305:537, (1983), the disclosure of which isincorporated herein by reference). Similar procedures for generatingheterospecific antibodies are disclosed, e.g., in WO 93/08829; WO91/00360, WO 92/00373; EP 03089; U.S. Pat. Nos. 6,210,668; 6,193,967;6,132,992; 6,106,833; 6,060,285; 6,037,453; 6,010,902; 5,989,530;5,959,084; 5,959,083; 5,932,448; 5,833,985; 5,821,333; 5,807,706;5,643,759, 5,601,819; 5,582,996; 5,496,549; 4,676,980; as well as inTraunecker et al., EMBO J. 10:3655 (1991); and Suresh et al., Methods inEnzymology 121:210 (1986); the disclosures of each of which areincorporated herein by reference. Heterospecific antibodies can includeFc mutations that enforce correct chain association in multi-specificantibodies, as described, e.g., by Klein et al, mAbs 4(6):653-663,(2012); the disclosure of which is incorporated herein by reference.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, CL, CHdomains (e.g., C_(H)1, C_(H)2, C_(H)3), hinge, (V_(L), V_(H))) isderived from a human germline immonglobulin sequence. A human antibodycan be produced in a human cell (e.g., by recombinant expression), or bya non-human animal or a prokaryotic or eukaryotic cell that is capableof expressing functionally rearranged human immunoglobulin (e.g., heavychain and/or light chain) genes. Human antibodies can be made by avariety of methods known in the art including phage display methodsusing antibody libraries derived from human immunoglobulin sequences.See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO1998/46645; WO 1998/50433; WO 1998/24893; WO 1998/16654; WO 1996/34096;WO 1996/33735; and WO 1991/10741; the disclosure of each of which isincorporated herein by reference. Human antibodies can also be producedusing transgenic mice that are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes. See, e.g., PCT publications WO 98/24893; WO 92/01047; WO96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425;5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and5,939,598; the disclosure of each of which is incorporated by referenceherein.

As used herein, the term “humanized” antibodies refers to forms ofnon-human (e.g., primate, murine, rabbit, goat, rodent, or othernon-human mammal) antibodies that are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other target-binding subdomains of antibodies) which containminimal sequences derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin. Allor substantially all of the FR regions may also be those of a humanimmunoglobulin sequence. The humanized antibody can also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin consensus sequence. Methods of antibodyhumanization are known in the art. See, e.g., Riechmann et al., Nature332:323-7, 1988; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761;5,693,762; and U.S. Pat. No. 6,180,370 to Queen et al; EP239400; PCTpublication WO 91/09967; U.S. Pat. No. 5,225,539; EP592106; andEP519596; incorporated herein by reference.

As used herein, the term “monoclonal antibody” refers to an antibodythat is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

As used herein, the term “multi-specific antibodies” refers toantibodies that exhibit affinity for more than one target antigen.Multi-specific antibodies can have structures similar to fullimmunoglobulin molecules and include Fc regions, for example IgG Fcregions. Such structures can include, but not limited to, IgG-Fv,IgG-(scFv)₂, DVD-Ig, (scFv)₂-(scFv)₂-Fc and (scFv)₂-Fc-(scFv)₂. In caseof IgG-(scFv)₂, the scFv can be attached to either the N-terminal or theC-terminal end of either the heavy chain or the light chain. Exemplarymulti-specific molecules that include Fc regions and into which TNFR2antibodies or antigen-binding fragments thereof can be incorporated havebeen reviewed, e.g., by Kontermann, 2012, mAbs 4(2):182-197, Yazaki etal, 2013, Protein Engineering, Design & Selection 26(3):187-193, andGrote et al, 2012, in Proetzel & Ebersbach (eds.), Antibody Methods andProtocols, Methods in Molecular Biology vol. 901, chapter 16:247-263;incorporated herein by reference. In certain cases, antibody fragmentscan be components of multi-specific molecules without Fc regions, basedon fragments of IgG or DVD or scFv. Exemplary multi-specific moleculesthat lack Fc regions and into which antibodies or antibody fragments canbe incorporated include scFv dimers (diabodies), trimers (triabodies)and tetramers (tetrabodies), Fab dimers (conjugates by adhesivepolypeptide or protein domains) and Fab trimers (chemically conjugated),are described by Hudson and Souriau, 2003, Nature Medicine 9:129-134;incorporated herein by reference.

As used herein, the term “non-native constant region” refers to anantibody constant region that is derived from a source that is differentfrom that of the antibody variable region or that is a human-generatedsynthetic polypeptide having an amino sequence that is different fromthe native antibody constant region sequence. For instance, an antibodycontaining a non-native constant region may have a variable regionderived from a non-human source (e.g., a mouse, rat, or rabbit) and aconstant region derived from a human source (e.g., a human antibodyconstant region).

As used herein, the term “percent (%) sequence identity” refers to thepercentage of amino acid (or nucleic acid) residues of a candidatesequence that are identical to the amino acid (or nucleic acid) residuesof a reference sequence after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity(e.g., gaps can be introduced in one or both of the candidate andreference sequences for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). Alignment for purposes ofdetermining percent sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publiclyavailable computer software, such as BLAST, ALIGN, or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For example, a reference sequence aligned for comparison with acandidate sequence may show that the candidate sequence exhibits from50% to 100% sequence identity across the full length of the candidatesequence or a selected portion of contiguous amino acid (or nucleicacid) residues of the candidate sequence. The length of the candidatesequence aligned for comparison purposes may be, for example, at least30%, (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length ofthe reference sequence. When a position in the candidate sequence isoccupied by the same amino acid residue as the corresponding position inthe reference sequence, then the molecules are identical at thatposition.

As used herein, the term “primatized antibody” refers to an antibodycomprising framework regions from primate-derived antibodies and otherregions, such as CDRs and constant regions, from antibodies of anon-primate source. Methods for producing primatized antibodies areknown in the art. See e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and5,693,780; incorporated herein by reference.

As used herein, the term “operatively linked” in the context of apolynucleotide fragment is intended to mean that the two polynucleotidefragments are joined such that the amino acid sequences encoded by thetwo polynucleotide fragments remain in-frame.

As used herein, the term “pharmacokinetic profile” refers to theabsorption, distribution, metabolism, and clearance of a drug (e.g., anantibody) over time following administration of the drug to a patient.

As used herein, the term “regulatory sequence” includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals) that control the transcription or translation of the antibodychain genes. Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185 (AcademicPress, San Diego, Calif., 1990); incorporated herein by reference.

As used herein, the term “scFv” refers to a single chain Fv antibody inwhich the variable domains of the heavy chain and the light chain froman antibody have been joined to form one chain. scFv fragments contain asingle polypeptide chain that includes the variable region of anantibody light chain (V_(L)) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) andthe variable region of an antibody heavy chain (V_(H)) (e.g., CDR-H1,CDR-H2, and/or CDR-H3) separated by a linker. The linker that joins theV_(L) and V_(H) regions of a scFv fragment can be a peptide linkercomposed of proteinogenic amino acids. Alternative linkers can be usedto so as to increase the resistance of the scFv fragment to proteolyticdegradation (e.g., linkers containing D-amino acids), in order toenhance the solubility of the scFv fragment (e.g., hydrophilic linkerssuch as polyethylene glycol-containing linkers or polypeptidescontaining repeating glycine and serine residues), to improve thebiophysical stability of the molecule (e.g., a linker containingcysteine residues that form intramolecular or intermolecular disulfidebonds), or to attenuate the immunogenicity of the scFv fragment (e.g.,linkers containing glycosylation sites). scFv molecules are known in theart and are described, e.g., in U.S. Pat. No. 5,892,019; Flo et al.,(Gene 77:51, 1989); Bird et al., (Science 242:423, 1988); Pantoliano etal., (Biochemistry 30:10117, 1991); Milenic et al., (Cancer Research51:6363, 1991); and Takkinen et al., (Protein Engineering 4:837, 1991).The VL and VH domains of an scFv molecule can be derived from one ormore antibody molecules. It will also be understood by one of ordinaryskill in the art that the variable regions of the scFv molecules of theinvention can be modified such that they vary in amino acid sequencefrom the antibody molecule from which they were derived. For example, inone embodiment, nucleotide or amino acid substitutions leading toconservative substitutions or changes at amino acid residues can be made(e.g., in CDR and/or framework residues). Alternatively or in addition,mutations are made to CDR amino acid residues to optimizeantigen-binding using art-recognized techniques. ScFv fragments aredescribed, for example, in WO 2011/084714; incorporated herein byreference.

As used herein, a small modular immunopharmaceutical (SMIP) proteinrefers to a protein that contains one or more of the followingimmunoglobulin domains: an antigen-binding domain, an immunoglobulinhinge region or a domain derived there from, an immunoglobulin heavychain C_(H)2 constant region or a domain derived there from, and animmunoglobulin heavy chain C_(H)3 constant region or a domain derivedthere from. Polypeptides containing one or more of these domains can beobtained using methods known in the art or described herein, e.g., byrecombinant expression of a polynucleotide encoding one or more of thesedomains or by chemical synthesis techniques (e.g., solid phase peptidesynthesis, see Solid Phase Peptide Synthesis, 2nd ed., 1984 The PierceChemical Co., Rockford, 111; the disclosure of which is incorporatedherein by reference in its entirety).

As used herein, the phrase “specifically binds” refers to a bindingreaction which is determinative of the presence of an antigen in aheterogeneous population of proteins and other biological molecules thatis recognized, e.g., by an antibody or antigen-binding fragment thereof,with particularity. An antibody or antigen-binding fragment thereof thatspecifically binds to an antigen will bind to the antigen or anepitope(s) thereof with a K_(D) of less than 100 nM (e.g., between 1 pMand 100 nM). An antibody or antigen-binding fragment thereof that doesnot exhibit specific binding to a particular antigen or epitope thereofwill exhibit a K_(D) of greater than 100 nM (e.g., greater than 500 nM,1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitopethereof. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein orcarbohydrate. For example, solid-phase ELISA immunoassays are routinelyused to select antibodies specifically immunoreactive with a protein orcarbohydrate. See, e.g., Harlow & Lane, Antibodies, A Laboratory Manual,Cold Spring Harbor Press, New York (1988); and Harlow & Lane, UsingAntibodies, A Laboratory Manual, Cold Spring Harbor Press, New York(1999), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

As used herein, the terms “subject” and “patient” refer to an organismthat receives treatment (e.g., by administration of an agonistic TNFR2antibody or antigen-binding fragment thereof of the invention) for aparticular disease or condition as described herein (such as animmunological disorder, e.g., an autoimmune disease). Examples ofsubjects and patients include mammals, such as humans, primates, pigs,goats, rabbits, hamsters, cats, dogs, guinea pigs, members of thebovidae family (such as cattle, bison, buffalo, and yaks, among others),cows, sheep, horses, and bison, among others, receiving treatment fordiseases or conditions, for example, immunological disorders, such asautoimmune disorders, graft-versus-host disease, allograft rejection,allergic reactions, and asthma, among others. A patient that is eligiblefor treatment with the compositions and methods of the invention mayhave an established disease (e.g., an established immunologicaldisorder, such as an autoimmune disease), in which case the patient hasbeen diagnosed as having the disease and has shown symptoms of thedisease for a prolonged period of time (e.g., over the course of days,weeks, months, or years). Alternatively, a patient may be symptomaticfor a particular disease, such as an immunological disorder describedherein, but has yet to be diagnosed with the disease by a physician.Other patients eligible for treatment with the compositions and methodsof the invention include those that have been diagnosed as having animmunological disorder, and may or may not be showing symptoms of thedisease as of yet. For example, a patient eligible for treatment withthe compositions and methods of the invention may be described asdiagnosed but asymptomatic if the patient has received a diagnosis of animmunological disorder, such as multiple sclerosis, e.g., by detectionof depleted myelin sheath around one or more neurons of the patient dueto the activity of autoreactive T-cells, even though the patient may notyet be showing symptoms of multiple sclerosis (e.g., lack of balance,reduced cognitive performance, blurred vision, or attenuatedcoordination, among others). Another example of a patient that has beendiagnosed with an immunological condition but is asymptomatic as of yetincludes a patient that has been diagnosed with rheumatoid arthritis,e.g., by the detection of autoreactive T-cells in a lymph sampleisolated from the patient, even though the patient has not yet presentedwith the symptoms associated with this disease, such as joint pain,joint stiffness, and a decrease in the muscle range or movement, amongothers.

As used herein, the terms “tumor necrosis factor receptor superfamily,”“TNFR superfamily,” or “TNFRSF” refer to a group of type I transmembraneproteins, with a carboxy-terminal intracellular domain and anamino-terminal extracellular domain characterized by a common cysteinerich domain (CRD). The TNFR superfamily includes receptors that mediatecellular signaling as a consequence of binding to one or more ligands inthe TNF superfamily. The TNFR superfamily can be divided into twosubgroups: receptors containing the intracellular death domain and thoselacking this domain. The death domain is an 80 amino acid motif thatpropagates apoptotic signal transduction cascades following receptoractivation. Exemplary TNFR super family members that contain theintracellular death domain include TNFR1, while TNFR2 represents a TNFRsuper family protein that does not contain this domain. Members of theTNFR superfamily include TNFR1, TNFR2, RANK, CD30, CD40, Lymphotoxinbeta receptor (LT-PR), OX40, Fas receptor, Decoy receptor 3, CD27, 4-1BB, Death receptor 4, Death receptor 5, Decoy receptor 1, Decoy receptor2, Osteoprotegrin, TWEAK receptor, TACI, BAFF receptor, Herpesvirusentry mediator, Nerve growth factor receptor, B-cell maturation antigen,Glucocorticoid-induced TNFR-related, TROY, Death receptor 6, Deathreceptor 3, and Ectodysplasin A2 receptor.

As used herein, the term “transfection” refers to any of a wide varietyof techniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation, heat shock,lipofection, calcium phosphate precipitation, DEAE-dextran transfectionand the like.

As used herein, the terms “treat” or “treatment” refer to therapeutictreatment, in which the object is to inhibit or slow down (lessen) anundesired physiological change or disorder, such as an immunologicaldisorder (e.g., autoimmune disease, allergic reaction, graft-versus-hostdisease, or allograft rejection). Beneficial or desired clinical resultsof treatment include, without limitation, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. Those in need of treatmentinclude those already with the condition or disorder, as well as thoseprone to have the condition or disorder or those in which the conditionor disorder is to be inhibited.

As used herein the term “variable region CDR” includes amino acids in aCDR or complementarity determining region as identified using sequence-or structure-based methods. As used herein, the term “CDR” or“complementarity determining region” refers to the noncontiguousantigen-binding sites found within the variable regions of both heavyand light chain polypeptides. These particular regions have beendescribed by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat,et al., Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242, 1991; Chothia et al., J. Mol. Biol. 196:901-917 (1987); and byMacCallum et al., J. Mol. Biol. 262:732-745 (1996); the disclosures ofeach of which are incorporated herein by reference. For agonistic TNFR2antibodies of the invention, a CDR, as defined by Kabat, may be based onsequence comparisons.

As used herein, the term “vector” includes a nucleic acid vector, e.g.,a DNA vector, such as a plasmid, a RNA vector, virus or other suitablereplicon (e.g., viral vector). A variety of vectors have been developedfor the delivery of polynucleotides encoding exogenous proteins into aprokaryotic or eukaryotic cell. Examples of such expression vectors aredisclosed in, e.g., WO 1994/11026, the disclosure of which isincorporated herein by reference. Expression vectors of the inventioncontain a polynucleotide sequence, as well as, e.g., additional sequenceelements used for the expression of proteins and/or the integration ofthese polynucleotide sequences into the genome of a cell, such as amammalian cell (e.g., a human cell). Vectors that can be used for theexpression of antibodies and antibody fragments of the invention includeplasmids that contain regulatory sequences, such as promoter andenhancer regions, which direct gene transcription. Other useful vectorsfor expression of antibodies and antibody fragments containpolynucleotide sequences that enhance the rate of translation of thesegenes or improve the stability or nuclear export of the mRNA thatresults from gene transcription. These sequence elements include, e.g.,5′ and 3′ untranslated regions, an internal ribosomal entry site (IRES),and polyadenylation signal site in order to direct efficienttranscription of the gene carried on the expression vector. Theexpression vectors of the invention may also contain a polynucleotideencoding a marker for selection of cells that contain such a vector.Examples of a suitable marker include genes that encode resistance toantibiotics, such as ampicillin, chloramphenicol, kanamycin, ornourseothricin.

As used herein, the term “VH” refers to the variable region of animmunoglobulin heavy chain of an antibody, including, e.g., the heavychain of an Fv, scFv, Fab, F(ab′)₂, Fd, scFv, SMIP, diabody, triabody,affibody, or nanobody. References to “VL” refer to the variable regionof an immunoglobulin light chain, including, e.g., the light chain of anFv, scFv, Fab, F(ab′)₂, Fd, scFv, SMIP, diabody, triabody, affibody, ornanobody. Antibodies and immunoglobulins are glycoproteins having thesame structural characteristics. While antibodies exhibit bindingspecificity to a specific target, immunoglobulins include bothantibodies and other antibody-like molecules which lack targetspecificity. Native antibodies and immunoglobulins are typicallyheterotetrameric glycoproteins of about 150,000 Daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachheavy chain of a native antibody has at the amino terminus a variabledomain (VH) followed by a number of constant domains. Each light chainof a native antibody has a variable domain at the amino terminus (VL)and a constant domain at the carboxy terminus.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B is the amino acid sequence of human TNFR2 (SEQ ID NO:366). Notably, human TNFR2 is numbered herein starting with anN-terminal methionine at position 1 and concluding with a C-terminalserine at position 461. All references to amino acid positions withinTNFR2 are made in the context of the TNFR2 numbering scheme shown inFIGS. 1A and 1B and recited in SEQ ID NO: 366. (FIG. 1A) Shaded residuesKCSPG (SEQ ID NO: 367) define an epitope within TNFR2 that isspecifically bound by the agonistic TNFR2 antibody MR2-1. MR2-1additionally binds an epitope that includes shaded residues SSDQVET (SEQID NO: 368) and an epitope that includesTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA (SEQ ID NO: 370). Though theseresidues are not consecutive in primary sequence with the KCSPG motif,they are spatially proximal in the three dimensional tertiary structureof TNFR2 and, for MR2-1, may form a discontinuous epitope that isappropriately positioned for interaction with other agonisticTNFR2antibodies of the invention (see FIG. 4). Agonistic TNFR2 antibodies ofthe invention bind the KCSPG epitope and may bind one or more of theresidues within these other regions. (FIG. 1B) Agonistic TNFR2 antibody8E6.D1 binds an epitope containing the KCSPG motif within TNFR2 (shownas shaded residues). Significantly, agonistic 8E6.D.1 does not bind anepitope containing the KCRPG motif (SEQ ID NO: 375), shown as underlinedresidues.

FIG. 2 is a table showing the raw data obtained from enzyme-linkedimmunosorbant assay (ELISA) experiments that were conducted to determinethe affinity of agonistic TNFR2 antibody MR2-1 for various continuousand discontinuous epitopes within TNFR2 (see Example 1). Rawluminescence values are shown in the fourth column of the table (right).Peptide sequences represent those that contain a portion of aconformational epitope within TNFR2 that interacts with MR2-1. Aminoacid residues with the single-digit code “2” designate cysteine residuesthat were chemically protected at the thiol position with anacetamidomethyl (ACM) moiety. These residues are not reactive withbromomethyl-containing electrophiles and were therefore not cross-linkedduring the cyclization and bicyclization phases of peptide synthesis.The third column in the table indicates the general structure of thepeptide scaffold. “LIN” indicates a linear 15-residue peptide that wasnot subject to an intramolecular cross-linking reaction. “LIN.AA”indicates a linear 15-residue peptide equivalent to the “LIN” group,except residues at positions 10 and 11 of the peptide were substitutedwith alanine. Where alanine occurred at these positions in the nativeTNFR2 sequence, these residues were substituted with glycine. “LOOP”indicates a 17-residue peptide in which cysteine residues were insertedat positions 1 and 17 of the peptide chain and were cross-linked byreaction with 2,6-bis(bromomethyl)pyridine. “LOOP.AA” indicates apeptide that is equivalent to the “LOOP” group, except residues atpositions 12 and 13 were substituted with alanine. Where alanineoccurred at these positions in the native TNFR2 sequence, these residueswere substituted with glycine. “MAT” indicates a peptide in whichcysteine residues were inserted at positions 1, 17, and 33 and weresubsequently cross-linked by reaction with1,3,5-bis(bromomethyl)benzene. Positions 2-16 and 18-32 represent15-residue peptides derived from TNFR2. “BET1” indicates a 24-residuepeptide derived from TNFR2. Cys residues were inserted into thesepeptides at positions 1 and 24 and were subsequently cross-linked byreaction with 2,6-bis(bromomethyl)pyridine. Proline and glycine wereincorporated into these peptides at positions 9 and 10 in order tonucleate a β-turn, and native cysteine residues were substituted withalanine residues. “BET2” indicates an 18-residue peptide derived fromTNFR2. Cys residues were inserted into these peptides at positions 1 and18 and were subsequently cross-linked by reaction with2,6-bis(bromomethyl)pyridine. Proline and glycine were incorporated intothese peptides at positions 9 and 10 in order to nucleate a β-turn, andnative cysteine residues were substituted with alanine residues. “CYS.S”indicates a 27-residue peptide in which positions 1-11 and 17-27represent 11-residue peptides derived from TNFR2 that contain cysteineresidues that form disulfide bridges in the native protein based oninformation available for UniProt entry P20333. The sequenceGly-Gly-Ser-Gly-Gly was incorporated into positions 12-16 of peptides ofthis group. Native Cys residues that do not form disulfide bridges wereprotected with acetamidomethyl (ACM) protecting groups and aredesignated with the single-digit code “2”. “CYS.D” indicates a22-residue peptide derived from TNFR2 that contains cysteine residuesthat form disulfide bridges in the native protein based on informationavailable for UniProt entry P20333. Native Cys residues that do not formdisulfide bridges were protected with acetamidomethyl (ACM) protectinggroups and are designated with the single-digit code “2”.

FIG. 3 is a table showing the results of an ELISA-based assay used toscreen a series of linear peptides derived from the human TNFR2 sequencefor those that bind agonistic antibody MR2-1 with high affinity. Columntwo of the table shows the positions within the human TNFR2 sequencefrom which the synthetic peptides were derived. The relative affinity ofeach of the screened linear peptides is shown in the fourth column ofthe table, and raw luminescence values are provided in column five.

FIG. 4 is a schematic illustrating the conformational epitopes withinTNFR2 that may interact with agonistic TNFR2 antibodies of theinvention, as well as residues that do not interact with particularagonistic TNFR2 antibodies. The KCSPG motif is shown in the expansion atthe top left of the figure; the KCRPG motif is shown in the expansion atthe right of the figure. Exterior surface of the protein designates thevan der Waals surface of TNFR2. FIG. 4 is a rendering of a monomer ofTNFR2 isolated from the X-ray crystal structure of TNFR2 (PDB ID: 3ALQ,Mukai, et al., Sci. Signal., 3:ra83 (2010)).

FIG. 5 is a graph showing the results of a T-reg induction assayconducted in order to determine the effect of agonistic TNFR2 antibodyMR2-1 on the proliferation of T-reg cells. Values shown on the y-axisrepresent the percent change in the quantity of cultured T-reg cells asa function of MR2-1 concentration (shown in pg/mL). This experimentdemonstrates a dose-dependent ability of agonistic TNFR2 antibodies toinduce the expansion of T-reg cells.

FIGS. 6A and 6B are graphs showing the results of T-reg induction assaysconducted in order to determine the effect of agonistic TNFR2 antibodieson the proliferation of T-reg cells. FIG. 6A is a graph showing theeffect of antibody MR2-1 on the induction of T-reg cell growth. Valuesshown on the y-axis represent the percent change in the quantity ofcultured T-reg cells over a 48 hour period in response to incubation ofpopulations of these cells with various external agents. Shown on theleft of the graph are the effects of IL-2, TNF, MR2-1, M1 (a negativecontrol that does not bind TNFR2), and MAB726 (a negative control thatfunctions as an antagonist of TNFR2). Bars shown in the center of thefigure demonstrate the effect of increasing 8E6-D1 titer on theproliferation of T-reg cells, as this agonistic TNFR2 antibody iscapable of inducing T-reg expansion in a dose-dependent fashion. Thebars on the right of the chart illustrate the effect of co-incubation of8E6-D1 and TNF on T-reg induction. FIG. 6B is a graph showing the effectof antibody 8E6.D1 on the induction of T-reg cell growth. Values shownon the y-axis represent the percent change in the quantity of culturedT-reg cells, as measured by FACS analysis, over a 48 hour period inresponse to incubation of populations of these cells with variousexternal agents. Shown on the left of the graph are the effects of IL-2,TNF, MR2-1, M1, and MAB726. Bars shown in the center of the figuredemonstrate the effect of increasing 8E6-D1 titer on the proliferationof T-reg cells, as this agonistic TNFR2 antibody is capable of inducingT-reg expansion in a dose-dependent fashion. The bars on the right ofthe chart illustrate the effect of co-incubation of 8E6-D1 and TNF onT-reg induction. Taken together, these data demonstrate the agonisticTNFR2 antibody 8E6-D1 is not only capable of inducing T-reg expansion,but can also synergize with the cognate TNFR2 ligand, TNF, to promoterobust T-reg proliferation.

DETAILED DESCRIPTION

Agonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention potentiate the activation of TNFR2 by binding this receptor(e.g., on the exterior surface of a T-reg cell) and inducing aTNFR2-mediated signal transduction cascade. Agonistic TNFR2 antibodiesmay promote TNFR2 signaling by nucleating a trimer of TNFR2 proteins atthe T-reg cell surface. This is the spatial configuration induced bybinding of TNFR2 to its cognate ligand, TNFα. This trimerization eventbrings individual TNFR2 proteins into close proximity and initiatessignaling via the MAPK/NFκB/TRAF2/3 pathway, which ultimately leads tocell growth and escape from apoptosis. Agonistic TNFR2 antibodies of theinvention may emulate the TNFR2-TNFα interaction by binding the receptorand triggering this structural change.

Antibodies or antigen-binding fragments thereof of the invention can beused to promote the proliferation of a population of T-reg cells andthus enhance the immunomodulatory activity of these cells. AgonisticTNFR2 antibodies and antigen-binding fragments thereof can therefore beused to attenuate an aberrant cell-mediated or humoral immune responseassociated with a variety of human diseases, such as autoimmunedisorders, asthma, allergic reactions, and diseases associated withallograft tolerance. For instance, agonistic TNFR2 antibodies of theinvention may be administered to suppress cytotoxic T-cell and B-cellactivity, thereby attenuating the response of a patient to a self orbenign antigen. Agonistic TNFR2 antibodies and antigen-binding fragmentsthereof can be administered to a mammalian subject, such as a human(e.g., by any of a number of routes of administration described herein)in order to attenuate an aberrant immune response, such as a responseagainst a self or non-threatening antigen. Alternatively, agonisticTNFR2 antibodies of the invention can be used to expand a population ofT-reg cells ex vivo that have been extracted, e.g., from a patient or anMHC-matched donor. After inducing proliferation of these T-reg cells inculture by treatment with an agonistic TNFR2 antibody or antigen-bindingfragment thereof of the invention, these cells can subsequently beadministered to a patient, e.g., using standard cellular administrationtechniques known in the art or described herein. In this way, agonisticTNFR2 antibodies of the invention may synergize with existing techniquesto suppress humoral and cell-mediated immune responses as a treatmentmodality for patients suffering from a variety of immunologicaldisorders.

Agonistic TNFR2 Antibodies

Agonistic TNFR2 antibodies of the invention include antigen-bindingfragments, such as an scFv, Fab, F(ab′)2, diabody, triabody, orantibody-like scaffold protein as described above, and may be of anyimmunoglobulin subtype, such as IgG, IgM, IgA, IgD, and IgE. Theanti-TNFR2 antibodies of the invention are capable of interacting withand promoting signal transduction events mediated by TNFR2. Thus, theTNFR2 antibodies of the invention can selectively potentiateTNFR2-mediated T-reg cell growth. Without being limited to anyparticular mechanism, this phenotype may be achieved due to the abilityof an agonistic TNFR2 antibody or antigen-binding fragment thereof ofthe invention to induce conformational changes within TNFR2 that lead toreceptor trimerization. This spatial configuration has been shown torender TNFR2 active for MAPK/TRAF 2/3 signal transduction, whichsubsequently leads to activation of NFκB-mediated transcription of genesinvolved in T-reg cell growth and escape from apoptosis (Faustman, etal., Nat. Rev. Drug Disc., 9:482-493 (2010), the disclosure of which isincorporated herein by reference).

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention may be capable of inducing the proliferation of a populationof T-reg cells, (e.g., levels of CD4+, CD25+, FOXP3+ T cells) e.g., by0.00001 to 100.0% (e.g., 0.00001%, 0.00002%, 0.00003%, 0.00004%,0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%,0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%,0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%,0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%,6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%,80.0%, 90.0%, or 100%), e.g., in vivo in a subject administered theantibody or antigen-binding fragment thereof, or in vitro in a samplecontaining the T-reg cells that are contacted with the antibody orantigen-binding fragment thereof, as measured, e.g., by FACS analysis,relative to a subject or sample containing a population of cells nottreated with an agonistic TNFR2 antibody or fragment thereof of theinvention.

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention can therefore be used to promote T-reg cell proliferation andcan be administered to a mammalian subject, such as a human patient,with an autoimmune disorder, in order to attenuate the magnitude andduration of an immune response (e.g., quantity of CD8+ cytotoxic Tlymphocytes produced in vivo in response to a self or non-threateningforeign antigen) in the patient. For instance, administration of anagonistic TNFR2 antibody or antigen-binding fragment thereof of theinvention to a human patient, or a population of T-reg cells expanded exvivo by treatment with the antibody or antigen-binding fragment thereofof the invention, can cause a reduction in the amount of secretedimmunoglobulin (e.g., IgG) that is cross-reactive with a self ornon-threatening antigen, e.g., by 0.00001 mg/mL to 10.0 mg/mL (e.g.,0.00001 mg/mL, 0.0001 mg/mL, 0.001 mg/mL, 0.01 mg/mL, 0.1 mg/mL, 1.0mg/mL, or 10.0 mg/mL), or by 0.001 to 1.0 mg/mL (e.g., 0.001 mg/mL,0.005 mg/mL, 0.010 mg/mL, 0.050 mg/mL, 0.10 mg/mL, 0.20 mg/mL, 0.30mg/mL, 0.40 mg/mL, 0.50 mg/mL, 0.60 mg/mL, 0.70 mg/mL, 0.80 mg/mL, 0.90mg/mL, or 1.0 mg/mL) relative to a subject not treated with an agonisticTNFR2 antibody or antigen-binding fragment thereof of the invention.Additionally or alternatively, agonistic TNFR2 antibodies orantigen-binding fragments thereof of the invention may be capable ofdiminishing cytotoxic T-cell counts (e.g., levels of CD8+ T cells) e.g.,by 0.00001 to 100.0% (e.g., 0.00001%, 0.00002%, 0.00003%, 0.00004%,0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%,0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%,0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%,0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%,6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%,80.0%, 90.0%, or 100%) in a subject as measured, e.g., by FACS analysisrelative to a subject not treated with an agonistic TNFR2 antibody orantigen-binding fragment thereof of the invention. For instance, anagonistic TNFR2 antibody or antigen-binding fragment thereof of theinvention can be administered to a subject (e.g., a mammalian subject,such as a human) in order to treat, e.g., an immunological diseasedescribed herein. Treatment of a subject in this manner may reduce thequantity of autoreactive CD8+ T-cells within the subject.

Agonistic TNFR2 antibodies of the invention may additionally attenuatethe secretion of IFNγ, an immunostimulatory cytokine, in a subject,e.g., by 0.00001 mg/mL to 10.0 mg/mL (e.g., 0.00001 mg/mL, 0.0001 mg/mL,0.001 mg/mL, 0.01 mg/mL, 0.1 mg/mL, 1.0 mg/mL, or 10.0 mg/mL), or by0.001 to 1.0 mg/mL (e.g., 0.001 mg/mL, 0.005 mg/mL, 0.010 mg/mL, 0.050mg/mL, 0.10 mg/mL, 0.20 mg/mL, 0.30 mg/mL, 0.40 mg/mL, 0.50 mg/mL, 0.60mg/mL, 0.70 mg/mL, 0.80 mg/mL, 0.90 mg/mL, or 1.0 mg/mL) relative to asubject not treated with an agonistic TNFR2 antibody of the invention.

Additionally or alternatively, agonistic TNFR2 antibodies orantigen-binding fragments thereof of the invention may be capable ofstimulating the transcription of various genes. For instance, anagonistic TNFR2 antibody or antigen-binding fragment thereof may inducethe expression of one or more of cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt,genes encoding proteins involved in the angiogenic pathway, IKKcomplexes, RIP, NIK, MAP3K, genes encoding proteins involved in the NFkBpathway, NIK, JNK, AP-1, a MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R,Foxp3, IL2, TNF, and lymphotoxin (e.g., lymphotoxin α and lymphotoxinβ). For instance, an agonistic TNFR2 antibody or antigen-bindingfragment thereof of the invention may be capable of inducing thetranscription of one or more of these genes, thus resulting in anincrease in the level of the mRNA transcripts derived from the one ormore genes and/or an increase in the level of protein encoded by the oneor more genes. The increase in expression of these genes can be detectedusing established molecular biology techniques known in the art, e.g.,by detecting an increase in mRNA levels by Northern blot analysis orreverse-transcription PCT (RT-PCR) methods, or by detecting an increasein protein levels by immunoblot analysis or ELISA-based techniques.Additionally or alternatively, an agonistic TNFR2 antibody orantigen-binding fragment thereof of the invention may be capable ofpromoting the activity of one or more proteins associated with the TNFR2signal transduction cascade (or related signaling pathways that areactivated as a result of TNFR2 signaling). For instance, an agonisticTNFR2 antibody or antigen-binding fragment thereof of the invention maybe capable of promoting an increase in the phosphorylation of one ormore proteins, such as cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, genesencoding proteins involved in the angiogenic pathway, IKK complexes,RIP, NIK, MAP3K, genes encoding proteins involved in the NFkB pathway,NIK, JNK, AP-1, a MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R, Foxp3, IL2,TNF, and lymphotoxin (e.g., lymphotoxin α and lymphotoxin β). Anincrease in the phosphorylation of one or more proteins that occurs,e.g., as a result of treatment of a subject or of a sample of cellsisolated from a subject can be detected using standard molecular biologytechniques known in the art, such as by immunoblot analysis orELISA-based techniques.

Agonistic TNFR2 antibodies of the invention are capable ofdiscriminating among the members of the tumor necrosis factor receptorsuperfamily (TNFRSF). Preferred agonistic TNFR2 antibodies andantigen-binding fragments thereof are those that do not specificallybind a TNFRSF member other than TNFR2. The TNFR superfamily includesreceptors that mediate cellular signaling as a consequence of binding toone or more ligands in the TNF superfamily. The TNFR superfamily can bedivided categorically into two types of receptors on the basis ofwhether the receptor contains an intracellular death domain, an 80-aminoacid motif that propagates apoptotic signal transduction cascadesfollowing receptor activation. Exemplary TNFR super family members thatcontain the intracellular death domain include TNFR1, while TNFR2represents a TNFR super family protein that does not contain thisdomain. Members of the TNFR superfamily include TNFR1, TNFR2, RANK,CD30, CD40, Lymphotoxin beta receptor (LT-8R), OX40, Fas receptor, Decoyreceptor 3, CD27, 4-1 BB, Death receptor 4, Death receptor 5, Decoyreceptor 1, Decoy receptor 2, Osteoprotegrin, TWEAK receptor, TACI, BAFFreceptor, Herpesvirus entry mediator, Nerve growth factor receptor,B-cell maturation antigen, Glucocorticoid-induced TNFR-related, TROY,Death receptor 6, Death receptor 3, and Ectodysplasin A2 receptor.

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention can be assessed to determine whether they lack specificbinding for another TNFR superfamily member using a variety of in vitrobinding assays, such as ELISA-based methods. For instance, agonisticTNFR2 antibodies or antigen-binding fragments thereof of the inventionmay specifically bind human TNFR2 or a TNFR2-derived peptide, such asthe peptide fragment defined by residues 48-67 of SEQ ID NO: 366 withinhuman TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ 10 NO: 346), with an affinitythat is, e.g., at least 5-fold greater (e.g., 5-fold greater, 6-foldgreater, 7-fold greater, 8-fold greater, 9-fold greater, 10-foldgreater, 20-fold greater, 30-fold greater, 40-fold greater, 50-foldgreater, 60-fold greater, 60-fold greater, 70-fold greater, 80-foldgreater, 90-fold greater, 100-fold greater, 200-fold greater, 300-foldgreater, 400-fold greater, 500-fold greater, 600-fold greater, 700-foldgreater, 800-fold greater, 900-fold greater, 1,000-fold greater,2,000-fold greater, 3,000-fold greater, 4,000-fold greater, 5,000-foldgreater, 6,000-fold greater, 7,000-fold greater, 8,000-fold greater,9,000-fold greater, 10,000-fold greater, or more) than the affinity ofthe same antibody for another TNFR superfamily member. For instance,agonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention may bind the peptide defined by SEQ ID NO: 346 with anaffinity that is, e.g., 5-fold greater. 6-fold greater, 7-fold greater,8-fold greater, 9-fold greater, 10-fold greater, 20-fold greater,30-fold greater, 40-fold greater, 50-fold greater, 60-fold greater,60-fold greater, 70-fold greater, 80-fold greater, 90-fold greater,100-fold greater, 200-fold greater, 300-fold greater, 400-fold greater,500-fold greater, 600-fold greater, 700-fold greater, 800-fold greater,900-fold greater, 1,000-fold greater, 2,000-fold greater, 3,000-foldgreater, 4,000-fold greater, 5,000-fold greater, 6,000-fold greater,7,000-fold greater, 8,000-fold greater, 9,000-fold greater, or10,000-fold greater than the affinity of the same antibody for anotherTNFR superfamily member, such as TNFR1, RANK, CD30, CD40, Lymphotoxinbeta receptor (LT-PR), OX40, Fas receptor, Decoy receptor 3, CD27, 4-1BB, Death receptor 4, Death receptor 5, Decoy receptor 1, Decoy receptor2, Osteoprotegrin, TWEAK receptor, TACI, BAFF receptor, Herpesvirusentry mediator, Nerve growth factor receptor, B-cell maturation antigen,Glucocorticoid-induced TNFR-related, TROY, Death receptor 6, Deathreceptor 3, or Ectodysplasin A2 receptor.

Additionally, agonistic TNFR2 antibodies and antigen-binding fragmentsof the invention may, but preferably do not, bind an epitope containingresidues 142-146 of SEQ ID NO: 366 within human TNFR2 (KCRPG, SEQ ID NO:375). For instance, agonistic TNFR2 antibodies of the invention may bindan epitope containing residues 130-149 of SEQ ID NO: 366(KQEGCRLCAPLRKCRPGFGV, SEQ ID NO: 357).

Preferred agonistic TNFR2 antibodies and antigen-binding fragments ofthe invention are those that specifically bind the KCSPG epitope ofTNFR2, but do not specifically bind the KCRPG epitope of TNFR2 (i.e.,the agonistic TNFR2 antibodies and antigen-binding fragments candistinguish between these two epitopes). For instance, an agonisticTNFR2 antibody or antigen-binding fragment of the invention may bind anepitope containing one or more residues within the KCSPG motif of TNFR2with an affinity that is, e.g., 5-fold greater, 6-fold greater, 7-foldgreater, 8-fold greater, 9-fold greater, 10-fold greater, 20-foldgreater, 30-fold greater, 40-fold greater, 50-fold greater, 60-foldgreater, 60-fold greater, 70-fold greater, 80-fold greater, 90-foldgreater, 100-fold greater, 200-fold greater, 300-fold greater, 400-foldgreater, 500-fold greater, 600-fold greater, 700-fold greater, 800-foldgreater, 900-fold greater, 1,000-fold greater, 2,000-fold greater,3,000-fold greater, 4,000-fold greater, 5,000-bold greater, 6,000-foldgreater, 7,000-fold greater, 8,000-fold greater, 9,000-fold greater, or10,000-fold greater than the affinity of the same antibody orantigen-binding fragment for a peptide that contains the KCRPG sequenceof human TNFR2 (SEQ ID NO: 375). For example, agonistic TNFR2 antibodiesand antigen-binding fragments of the invention may bind an epitopecontaining one or more residues of the KCSPG motif, such as the peptidedefined by residues 48-67 of human TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ IDNO: 346) with an affinity that is, e.g., 5-fold greater, 6-fold greater,7-fold greater, 8-fold greater, 9-fold greater, 10-fold greater, 20-foldgreater, 30-fold greater, 40-fold greater, 50-fold greater, 60-foldgreater, 60-fold greater, 70-fold greater, 80-fold greater, 90-foldgreater, 100-fold greater, 200-fold greater, 300-fold greater, 400-foldgreater, 500-fold greater, 600-fold greater, 700-fold greater, 800-foldgreater, 900-fold greater, 1,000-fold greater, 2,000-fold greater,3,000-fold greater, 4,000-fold greater, 5,000-fold greater, 6,000-foldgreater, 7,000-fold greater, 8,000-fold greater, 9,000-fold greater, or10,000-fold greater than the affinity of the same antibody orantigen-binding fragment for a peptide that contains the KCRPG sequence(SEQ ID NO: 375) of human TNFR2, such as a peptide defined by residues130-149 of SEQ ID NO: 366 (KQEGCRLCAPLRKCRPGFGV, SEQ ID NO: 357).

Specific Binding Properties of Agonistic TNFR2 Antibodies

The specific binding of an antibody or antibody fragment of theinvention to TNFR2 (e.g., human TNFR2) can be determined by any of avariety of established methods. The affinity can be representedquantitatively by various metrics, including the concentration ofantibody needed to achieve half-maximal potentiation of TNFR2 signallingin vitro (EC₅₀) and the equilibrium constant (K_(D)) of theantibody-TNFR2 complex dissociation. The equilibrium constant, K_(D),which describes the interaction of TNFR2 with an antibody orantigen-binding fragment thereof of the invention is the chemicalequilibrium constant for the dissociation reaction of a TNFR2-antibodycomplex into solvent-separated TNFR2 and antibody molecules that do notinteract with one another.

Antibodies of the invention are those that specifically bind to TNFR2with a K_(D) value of less than 100 nM (e.g., 95 nM, 90 nM, 85 nM, 80nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). Incertain cases, antibodies of the invention are those that specificallybind to TNFR2 with a K_(D) value of less than 1 nM (e.g., 990 pM, 980pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).

Antibodies of the invention can also be characterized by a variety of invitro binding assays. Examples of experiments that can be used todetermine the K_(D) or EC₅₀ of a TNFR2 antibody include, e.g., surfaceplasmon resonance, isothermal titration calorimetry, fluorescenceanisotropy, and ELISA-based assays, among others. ELISA represents aparticularly useful method for analyzing antibody activity, as suchassays typically require minimal concentrations of antibodies. A commonsignal that is analyzed in a typical ELISA assay is luminescence, whichis typically the result of the activity of a peroxidase conjugated to asecondary antibody that specifically binds a primary antibody (e.g., aTNFR2 antibody of the invention). Antibodies of the invention arecapable of binding TNFR2 and epitopes derived thereof, such as epitopescontaining one or more, or all, of residues 56-60 of SEQ ID NO: 366within human TNFR2 (KCSPG, SEQ ID NO: 367), as well as isolated peptidesderived from TNFR2 that structurally pre-organize various residues in amanner that may simulate the conformation of these amino acids in thenative protein. For instance, antibodies of the invention may bindpeptides containing one or more, or all, amino acids of this KCSPG motifand one or more additional amino acids of residues 48-67 of SEQ ID NO:366 within human TNFR2 (SEQ ID NO: 346). Such peptides may pre-disposeone or more residues of the KCSPG motif towards binding an agonisticTNFR2 antibody or antigen-binding fragment thereof, e.g., by selectivelypresenting a conformation of the KCSPG epitope that is similar to theconformation this epitope exhibits in native human TNFR2. Exemplarypeptides capable of stabilizing short sequence motifs include cyclic andbicyclic peptides, e.g., that feature an amide bond between the N- andC-terminal residues of the peptide and/or intramolecular cross-linksformed by reaction of a side-chain functional group (e.g., a cysteinethiolate) with a scaffolding molecule (e.g., a multivalent electrophile,such as 2,6-bis(bromomethyl)pyridine or 1,3,5-tris(bromomethyl)benzene).Other intramolecular cross-links, such as olefin-containing linkersformed by ring-closing metathesis, saturated alkyl linkers formed byolefin reduction, disulfide bridges formed by cysteine oxidation, andtriazole-containing linkers formed by azide-alkyne cycloadditionreactions are known in the art and can be used to produce a peptide thatrestricts the conformation of an epitope within TNFR2, such as the KCSPGmotif, to that which is presented in the native protein (see, e.g., WO2014/190257; WO 2008/040833; WO 2012/057624; U.S. Pat. Nos. 7,999,068;and 8,778,844; the disclosures of each of which are incorporated hereinby reference).

The binding of an agonistic TNFR2 antibody or antigen-binding fragmentthereof of the invention to TNFR2 or a constrained peptide containingone or more, or all, of the residues of the KCSPG motif can bequantified, e.g., via an ELISA-based technique. For instance, one candetermine whether a TNFR2 antibody binds to a particular epitope withinTNFR2 by analyzing the luminescence that occurs upon incubation of anHRP substrate (e.g., 2,2′-azino-di-3-ethylbenzthiazoline sulfonate) witha complex containing an antigen (e.g., a TNFR2-derived peptide) and aTNFR2 antibody when the complex is bound to a HRP-conjugated secondaryantibody. For instance, TNFR2 antibodies of the invention may induce aluminescence response of about 150 absorbance units or more whenincubated with surface-immobilized antigen and a HRP-conjugatedsecondary antibody in the presence of an HRP substrate. In certaincases, the luminescence observed can be from about 150 to about 1,400absorbance units (e.g., 200-1,000 absorbance units, 300-900 absorbanceunits, or 400-800 absorbance units). In particular cases, theluminescence observed can be from about 150 to about 300 absorbanceunits (e.g., 150-200 absorbance units or 200-250 absorbance units).

Agonistic TNFR2 Antibodies that Bind TNFR2 from Non-Human Animals

In addition to binding one or more, or all, of the residues of the KCSPGmotif, or polypeptides containing the KCSPG motif, agonistic TNFR2antibodies and antigen-binding fragments thereof of the invention alsoinclude those that specifically bind epitopes containing the equivalentTNFR2 motif of non-human animals, such as non-human mammals, e.g., in acow, bison, boar, mouse, or rat, among others. In these non-humanmammals, the TNFR2 sequence features a KCPPG motif (SEQ ID NO: 396) inplace of a KCSPG motif. Agonistic TNFR2 antibodies and antigen-bindingfragments of the invention also include those that are capable ofspecifically binding epitopes that contain one or more, or all, of theresidues KCPPG in TNFR2 derived from a non-human animal. These agonisticTNFR2 antibodies and antigen-binding fragments may be administered,e.g., to non-human mammals.

Sequences within non-human TNFR2 that are equivalent to the KCSPG motifin human TNFR2 are shown in Table 2 below.

TABLE 2 Location of sequences equivalent to KCSPG in TNFR2 fromnon-human mammals Amino acid positions of SEQ ID equivalent NO. ofGenbank Sequence sequence full-length Accession No. of Source ofequivalent within TNFR2 full-length TNFR2 to KCSPG TNFR2 sequence TNFR2sequence Human KCSPG 56-60 366 P20333.3 Cattle KCPPG 56-60 397 AAI05223Bison KCPPG 56-60 398 XP_010848145 Boar KCPPG 58-62 399 ABV02767.1 MouseKCPPG 57-61 400 AAA39752.1 Rat KCPPG 57-61 401 Q80WY6

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention may be capable of binding epitopes surrounding the KCPPG motifin TNFR2 derived from non-human animals. For instance, agonistic TNFR2antibodies and antigen-binding fragments thereof may be capable ofbinding epitopes such as QKIQMCCSKCPPGYRVQSLC in TNFR2 derived fromcattle (SEQ ID NO: 402), HKIQMCCSKCPPGYRVQSLC in TNFR2 derived frombison (SEQ ID NO: 403), TKAQMCCSKCPPGFRIQTSC in TNFR2 derived from boar(SEQ ID NO: 404), RKAQMCCAKCPPGQYVKHFC in TNFR2 derived from a mouse(SEQ ID NO: 405), and/or KKAQMCCAKCPPGQYAKHFC in TNFR2 derived from arat (SEQ ID NO: 406), as well as sequences that exhibit at least 85%sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequenceidentity) to these sequences and epitopes that contain conservativeamino acid substitutions relative to these sequences (e.g., so long asthe amino acids KCPPG are present in the epitope).

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention may be capable of binding epitopes surrounding downstream ofthe KCPPG motif in TNFR2 derived from non-human animals. For instance,agonistic TNFR2 antibodies and antigen-binding fragments thereof may becapable of binding epitopes such as SSDQVET in TNFR2 derived fromcattle, bison, and boar (SEQ ID NO: 368), TTDQVEI in TNFR2 derived frommice (SEQ ID NO: 407), and/or SDDQVET in TNFR2 derived from rats (SEQ IDNO: 408), as well as sequences that exhibit at least 85% sequenceidentity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) tothese sequences and epitopes that contain conservative amino acidsubstitutions relative to these sequences.

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention may be capable of binding additional epitopes downstream ofthe KCPPG motif in TNFR2 derived from non-human animals. For instance,agonistic TNFR2 antibodies and antigen-binding fragments thereof may becapable of binding epitopes such asTTKQNRICTCKPGWYCTLGRQEGCRLCVALRKCGPGFGVA in TNFR2 derived from cattleand bison (SEQ ID NO: 409), TPKQNRICSCKPGWYCTLGRQEGCRLCMALRKCSPGFGVT inTNFR2 derived from boar (SEQ ID NO: 410),TKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFGVA in TNFR2 derived from mice(SEQ ID NO: 411), and/or TKKQNRVCACNADSYCALKLHSGNCRQCMKLSKCGPGFGVA inTNFR2 derived from rats (SEQ ID NO: 412), as well as sequences thatexhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%,or 100% sequence identity) to these sequences and epitopes that containconservative amino acid substitutions relative to these sequences.

In certain cases, agonistic TNFR2 antibodies and antigen-bindingfragments thereof of the invention are capable of specifically bindingepitopes that contain one or more, or all of the residues KCPPG in TNFR2derived from a non-human animal and do not specifically bind a member ofthe TNFR superfamily other than TNFR2 (e.g., TNFR1, RANK, CD30, CD40,Lymphotoxin beta receptor (LT-PR), OX40, Fas receptor, Decoy receptor 3,CD27, 4-1 BB, Death receptor 4, Death receptor 5, Decoy receptor 1,Decoy receptor 2, Osteoprotegrin, TWEAK receptor, TACI, BAFF receptor,Herpesvirus entry mediator, Nerve growth factor receptor, B-cellmaturation antigen, Glucocorticoid-induced TNFR-related, TROY, Deathreceptor 6, Death receptor 3, and Ectodysplasin A2 receptor).

Additionally or alternatively, agonistic TNFR2 antibodies andantigen-binding fragments thereof of the invention for use in non-humanmammals are capable of specifically binding epitopes containing one ormore, or all, of the residues KCPPG in TNFR2 derived from a non-humananimal, but do not specifically bind an epitope in TNFR2 derived from anon-human animal containing a sequence equivalent to the KCRPG motifpresent in human TNFR2 (e.g., KCGPG or KCSPG, see Table 3 and sequencealignment below).

Sequences in TNFR2 derived from a non-human animal that are equivalentto the KCRPG motif in human TNFR2 include the KCGPG motif (SEQ ID NO:413) in cattle, bison, mice, and rats. The sequence in TNFR2 derivedfrom a boar that is equivalent to KCRPG found in human TNFR2 is KCSPG,located at amino acids 144-148 within boar TNFR2. Sequences within TNFR2derived from non-human animals that are equivalent to the KCRPG motif inhuman TNFR2 are shown in Table 3 below.

TABLE 3 Location of sequences equivalent to KCRPG in TNFR2 fromnon-human mammals Amino acid positions of SEQ ID equivalent NO. ofGenbank Sequence sequence full-length Accession No. of Source ofequivalent within TNFR2 full-length TNFR2 to KCRPG TNFR2 sequence TNFR2sequence Human KCRPG 142-146 366 P20333.3 Cattle KCGPG 142-146 397AAI05223 Bison KCGPG 142-146 398 XP_010848145 Boar KCSPG 144-148 399ABV02767.1 Mouse KCGPG 144-148 400 AAA39752.1 Rat KCGPG 144-148 401Q80WY6

Epitopes within TNFR2 derived from the non-human mammals discussed abovethat may be bound by agonistic TNFR2 antibodies and antigen-bindingfragments thereof of the invention are summarized in the sequencealignment below. This sequence alignment shows partial sequences ofTNFR2 derived from human, cattle, bison, boar, mouse, and rat, as wellas epitopes (shown in bold font) that may be bound by agonistic TNFR2antibodies and antigen-binding fragments thereof of the invention.

Alignment of partial TNFR2 sequences derived from human and select non-human mammalsHuman:   1MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRL--REYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSC 78 Cattle:   1MAPTAFWAALAVGLQFWAAGRAVPAQAVFTPYIPEPGSSCRQ--QEYYNQKIQMCCSKCPPGYRVQSLCNMTLDTICASC 78 Bison:   1MAPTAFWAALAVGLQFWAAGRAVPAQAVFTPYIPEPGSSCRQ--QEYYNHKIQMCCSKCPPGYRVQSLCNTTLDTICASC 78 Boar:   1MAPAAVWAALTVGLQLWAAGRAVPSQAVFMPYAPELGSSCRLPLKEYYDTKAQMCCSKCPPGFRIQTSCNRTSDTVCGSC 80 Mouse:   1MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQIS-QEYYDRKAQMCCAKCPPGQYVKHFCNKTSDTVCADC 79 Rat:   1MAPAALWVALVVELQLWATGHTVPAKVVLTPYKPEPGNQCQIS-QEYYDKKAQMCCAKCPPGQYAKHFCNKTSDTVCADC 79 Human:  79EDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEG-CRLCAPLRKCRPGFGVARPGTETS157 Cattle:  79ESSTYTQLWNLVTACFSCNSRCSSDQVETQACTTKQNRICTCKPGWYCTLGRQEG-CRLCVALRKCGPGFGVAKPGTATT157 Bison:  79ESSTYTQLWNLVTACFSCNSRCSSDQVETQACTTKQNRICTCKPGWYCTLGRQEG-CRLCVALRKCGPGFGVAKPGTATT157 Boar:  81ESSTYTQLWNSVSACFSCNSRCSSDQVETQACTPKQNRICSCKPGWYCTLGRQEG-CRLCMALRKCSPGFGVTKPGTATS159 Mouse:  80EASMYTQVWNQFRTCLSCSSSCTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGEGVASSRAPNG159 Rat:  80AAGMFTQVWNHLHTCLSCSSSCSDDQVETHNCTKKQNRVCACNADSYCALKLHSGNCRQCMKLSKCGPGFGVARSRTSNG159 Human: 158DVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCT------STSPTRSMAPGAVHLPQPVSTRSQHTQPTP231 Cattle: 158NVICAPCGPGTFSDTTSYTDTCKPHRNCSSVAIPGTASTDAVCT------SVLPTRKVARG------PATTRSQHMEPTL225 Bison: 158NVICAPCGPGTFSDTTSYTDTCKPHRNCSSVAIPGTASTDAVCT------SVLPTRKVARG------PATTRSQHMEPTL225 Boar: 160DVVCAPCAPGTFSSTLSSTDTCRPHRICSSVAIPGTARMDAVCT------SESPTLNVAQG------PAPTRSQRMEPTP227 Mouse: 160NVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDAVCAPESPTLSAIPR------TLYVSQPEPTRSQPLDQEP233 Rat: 160NVICSACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDAVCASESPTPSAVPR------TIYVSQPEPTRSQPMDQEP233 Human: 232EPSTAPSTSFLLPMGPSPPA----EGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADK307 Cattle: 226GPSTAPSTFFLLPKVPSPPSSPVEQPNTGNISLPIELIVGVTALGLLLIVVVNCVIMTQKKKKPFCLQGDAKVPHLPANK305 Bison: 226GPSTAPSTFFLLPKVPSPPSSPVEQPNAGNISLPIELIVGVTALGLLLIVVVNCVIMTQKKKKPFCLQGDAKVPHLPANK305 Boar: 228GPSVAPSTAPLPPMTPSPPSPPVEGLNTGNISLPIGLIVGVTAMGLLIIVLVNCVIMTQKKKKPFCLQGDAKVPHLPAKK307 Mouse: 234GPSQTPS---ILTSLGSTPI--IEQSTKGGISLPIGLIVGVTSLGLLMLGLVNCIILVQRKKKPSCLQRDAKVPHVPDEK308 Rat: 234GPSQTPH---IPVSLGSTPI--IEPSITGGISLPIGLIVGLTTLGLLMLGLANCFILVQRKKKPSCLQRETMVPHLPDDK308 Human: 308ARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVE-ASGAGEARASTGSSDSSPGGHGTQVNVTCIVN386 Cattle: 306AQGAPGPEQQHLLTTAPSSSSSSLESSTSSTDKRAPTRSQLQSPGVEKASTSGEAQTGCSSSEASSGGHGTQVNVTCIVN385 Bison: 306AQGAPGPEQQHLLTTAPSSSSSSLESSTSSTDKRAPTRSQLQSPGVE-ANTSGEAQTGCSSSEASSGGHGTQVNVTCIVN384 Boar: 308ARSVPGPEQQHLLTTAPSSSSSSLESSASAPDRRAPTPSQLQAPGADKTSGSGEARASSSSSESSSGSHGTQVNVTCIVN387 Mouse: 309SQDAVGLEQQHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVMAEAQGFQEARASSRISDSSHGSHGTHVNVTCIVN388 Rat: 309SQDAIGLEQQHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVTAEAQGSQEACAGSRSSDSSHGSHGTHVNVTCIVN388

Kinetic Properties of Agonistic TNFR2 Antibodies

In addition to the thermodynamic parameters of a TNFR2-antibodyinteraction, it is also possible to quantitatively characterize thekinetic association and dissociation of an antibody or antibody fragmentof the invention with TNFR2. This can be done by monitoring the rate ofantibody-antigen complex formation according to established procedures.For example, one can use surface plasmon resonance (SPR) to determinethe rate constants for the formation (k_(on)) and dissociation (k_(off))of an antibody-TNFR2 complex. These data also enable calculation of theequilibrium constant of (K_(D)) of antibody-TNFR2 complex dissociation,since the equilibrium constant of this unimolecular dissociation can beexpressed as the ratio of the k_(off) to k_(on) values. SPR is atechnique that is particularly advantageous for determining kinetic andthermodynamic parameters of receptor-antibody interactions since theexperiment does not require that one component be modified by attachmentof a chemical label. Rather, the receptor is typically immobilized on asolid metallic surface which is treated in pulses with solutions ofincreasing concentrations of antibody. Antibody-receptor binding inducesdistortion in the angle of reflection of incident light at the metallicsurface, and this change in refractive index over time as antibody isintroduced to the system can be fit to established regression modelsknown in the art in order to calculate the association and dissociationrate constants of an antibody-receptor interaction.

Antibodies of the invention exhibit high k_(on) and low k_(off) valuesupon interaction with TNFR2, consistent with high-affinity receptorbinding. For example, antibodies of the invention may exhibit k_(on)values in the presence of TNFR2 of greater than 10⁴ M⁻¹s⁻¹ (e.g.,1.0×10⁴ M⁻¹s⁻¹, 1.5×10⁴ M⁻¹s⁻¹, 2.0×10⁴ M⁻¹s⁻¹, 2.5×10⁴ M⁻¹s⁻¹, 3.0×10⁴M⁻¹s⁻¹, 3.5×10⁴ M⁻¹s⁻¹, 4.0×10⁴ s 4.5×10⁴ M⁻¹s⁻¹, 5.0×10⁴ M⁻¹s⁻¹,5.5×10⁴ M⁻¹s⁻¹, 6.0×10⁴ M⁻¹s⁻¹, 6.5×10⁴ M⁻¹s⁻¹, 7.0×10⁴ M⁻¹s⁻¹, 7.5×10⁴M⁻¹s⁻¹, 8.0×10⁴ M⁻¹s⁻¹, 8.5×10⁴ M⁻¹s⁻¹, 9.0×10⁴ M⁻¹s⁻¹, 9.5×10⁴ M⁻¹s⁻¹,1.0×10⁵ M⁻¹s⁻¹, 1.5×10⁵ M⁻¹s⁻¹, 2.0×10⁵ M⁻¹s⁻¹, 2.5×10⁵ M⁻¹s⁻¹, 3.0×10⁵M⁻¹s⁻¹, 3.5×10⁵ M⁻¹s⁻¹, 4.0×10⁵ M⁻¹s⁻¹, 4.5×10⁵ M⁻¹s⁻¹, 5.0×10⁵ M⁻¹s⁻¹,5.5×10⁵ M⁻¹s⁻¹, 6.0×10⁵ M⁻¹s⁻¹, 6.5×10⁵ M⁻¹s⁻¹, 7.0×10⁵ M⁻¹s⁻¹, 7.5×10⁵M⁻¹s⁻¹, 8.0×10⁵ M⁻¹s⁻¹, 8.5×10⁵ M⁻¹s⁻¹, 9.0×10⁵ M⁻¹s⁻¹, 9.5×10⁵ M⁻¹s⁻¹,or 1.0×10⁶ M⁻¹s⁻¹). Antibodies of the invention exhibit low k_(off)values when bound to TNFR2, since antibodies are capable of interactingwith distinct TNFR2 epitopes containing one or more of residues 56-60 ofSEQ ID NO: 366 within human TNFR2 with a high affinity. Epitopescontaining one or more of these residues form strong intermolecularcontacts with agonistic TNFR2 antibodies and antigen-binding fragmentsthereof of the invention, which serve to slow the dissociation of theantibody-TNFR2 complex. This high receptor affinity is manifested in lowk_(off) values. For instance, antibodies of the invention may exhibitk_(off) values of less than 10⁻³s⁻¹ when complexed to TNFR2 (e.g.,1.0×10⁻³s⁻¹, 9.5×10⁻⁴s⁻¹, 9.0×10⁻⁴s⁻¹, 8.5×10⁻⁴s⁻¹, 8.0×10⁻⁴s⁻¹,7.5×10⁻⁴s⁻¹, 7.0×10⁻⁴s⁻¹, 6.5×10⁻⁴ s⁻¹, 6.0×10⁻⁴s⁻¹, 5.5×10⁻⁴ s⁻¹,5.0×10⁻⁴s⁻¹, 4.5×10⁻⁴ s⁻¹, 4.0×10⁻⁴s⁻¹, 3.5×10⁻⁴s⁻¹, 3.0×10⁻⁴s⁻¹,2.5×10⁻⁴s⁻¹, 2.0×10⁻⁴s⁻¹, 1.5×10⁻⁴s⁻¹, 1.0×10⁻⁴s⁻¹, 9.5×10⁻⁵s⁻¹,9.0×10⁻⁵s⁻¹, 8.5×10⁻⁵s⁻¹, 8.0×10⁻⁵s⁻¹, 7.5×10⁻⁵s⁻¹, 7.0×10⁻⁵s⁻¹,6.5×10⁻⁵s⁻¹, 6.0×10⁻⁵s⁻¹, 5.5×10⁻⁵s⁻¹, 5.0×10⁻⁵s⁻¹, 4.5×10⁻⁵s⁻¹,4.0×10⁻⁵ s⁻¹, 3.5×10⁻⁵s⁻¹, 3.0×10⁻⁵ s⁻¹, 2.5×10⁻⁵s⁻¹, 2.0×10⁻⁵ s⁻¹,1.5×10⁻⁵ s⁻¹, or 1.0×10⁻⁵s⁻¹).

Epitopes within TNFR2 Bound by Agonistic TNFR2 Antibodies

The high affinities of antibodies of the invention for TNFR2 coupledwith the rapid onset of antibody-TNFR2 complex formation and the slowdissociation of these complexes render these antibodies well-suited fortherapeutic applications as activators of T-reg cell proliferation. Thehigh k_(on) values, for instance, indicate that antibodies of theinvention are capable of localizing to the surface of a TNFR2-expressingcell (e.g., a T-reg cell) and rapidly associating with TNFR2, therebyinducing receptor activation, e.g., in a fashion similar to that ofTNFα. Moreover, the slow dissociation of the antibody-TNFR2 complex canbe indicative of a long half-life of the complex in vivo, which resultsin stable, sustained up-regulation of the growth of the TNFR2-expressingcell (e.g., sustained up-regulation of T-reg growth, such as a CD4+,CD25+, FOXP3+ T-cell). These ideal thermodynamic and kinetic parametersof TNFR2 binding are consistent with the strong intermolecular contactsthat are established upon association of antibodies and antibodyfragments of the invention with TNFR2.

Among the difficulties in developing TNFR2 antibodies that are capableof inducing TNFR2 signal transduction stimulating the propagation ofT-reg cells has been the elucidation of epitopes within TNFR2 thatpromote agonistic receptor-binding. Particular, discrete peptidefragments found within the TNFR2 primary structure may bind agonisticantibodies of the invention by virtue of the spatial orientation ofthese residues in the native conformation of the receptor.Significantly, these residues have been difficult to identify, as manyisolated linear TNFR2-derived peptides do not appear to interact withagonistic TNFR2 antibodies due to the different conformations thesepeptides exhibit when structurally pre-organized within the full-lengthprotein and when isolated in solution. Epitope mapping analysis usinglinear peptides, as well as constrained cyclic and bicyclic peptides,derived from various regions of TNFR2 indicates that agonistic TNFR2antibodies of the invention bind epitopes from distinct regions of theTNFR2 amino acid sequence, and may bind these epitopes in aconformation-dependent manner. Particularly important epitopes that bindagonistic TNFR2 antibodies of the invention and promote receptoractivation are those that contain one or more, or all, of the residuesof the KCSPG motif (SEQ ID NO: 367), located at positions 56-60 of SEQID NO: 366 within human TNFR2. One or more of these residues may residewithin larger epitopes, such as residues 48-67 of SEQ ID NO: 366, whichmay interact with agonistic TNFR2 antibodies of the invention. Theknowledge of those residues that selectively bind TNFR2 antibodies in amanner that promotes receptor activation, and thus, T-reg cellproliferation, can be used to identify and design a wide array ofagonistic TNFR2 antibodies and antigen-binding fragments thereof usinglibrary screening techniques, such as those described herein or known inthe art. For instance, structurally rigidified peptides containing oneor more, or all, of the residues within the KCSPG sequence (e.g., acyclic or bicyclic peptide that presents the KCSPG motif in aconformation similar to that observed in native human TNFR2) can be usedto screen and select for antibodies, antigen-binding fragments, andantibody-like scaffolds that bind these epitopes with high affinity andselectivity.

Several distinct residues within TNFR2 bind agonistic TNFR2 antibodiesand antibody fragments of the invention and establish strongintermolecular contacts with these antibodies. Notably, functionalagonistic TNFR2 antibodies and antibody fragments of the inventionselectively bind an epitope containing one or more, or all, of aminoacids 56-60 of SEQ ID NO: 366 within human TNFR2 (KCSPG, SEQ ID NO:367). The spatial orientation of this epitope is shown in FIG. 4.Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention are capable of selectively binding an epitope of TNFR2 thatcontains one or more, or all, of the residues of the KCSPG motif withinTNFR2. For instance, antibodies of the invention may exhibit specificbinding to epitopes that include one or more, or all, of residues 48-67of SEQ ID NO: 366 within human TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO:346), as well as epitopes that exhibit at least 85% sequence identity(e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to thissequence and epitopes that contain conservative amino acid substitutionsrelative to this sequence (e.g., so long as the amino acids KCSPG arepresent in the epitope). The KCSPG sequence motif represents animportant functional epitope within TNFR2 towards promoting receptoractivation and initiating MAPK/NFκB/TRAF2/3 signaling. As such, theability of a TNFR2 antibody to interact with an epitope including one ormore, e.g., all, of residues 56-60 of SEQ ID NO: 366 within TNFR2characterizes antibodies of the invention that stimulate TNFR2 activity.

In addition to interacting with the KCSPG motif (SEQ ID NO: 367),agonistic TNFR2 antibodies of the invention may also specifically bindan epitope within human TNFR2 that includes at least five continuous ordiscontinuous residues from positions 96-154 of SEQ ID NO: 366 withinhuman TNFR2(CGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPSFGVARPGT, SEQ ID NO:371), as well as epitopes that exhibit at least 85% sequence identity(e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to thissequence and epitopes that contain conservative amino acid substitutionsrelative to this sequence.

Agonistic TNFR2 antibodies of the invention may also specifically bindan epitope containing at least five continuous or discontinuous residuesfrom positions 96-112 of SEQ ID NO: 366 within TNFR2 (CGSRCSSDQVETQACTR,SEQ ID NO: 372), or an epitope that exhibits at least 85% sequenceidentity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) tothis sequence or contains conservative amino acid substitutions relativeto this sequence. For example, in certain cases, agonistic TNFR2antibodies of the invention may specifically bind an epitope thatincludes one or more residues from positions 101-107 of SEQ ID NO: 366within TNFR2 (SSDQVET, SEQ ID NO: 368), as well as epitopes that exhibitat least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100%sequence identity) to this sequence and epitopes that containconservative amino acid substitutions relative to this sequence.

Additionally, agonistic TNFR2 antibodies of the invention mayspecifically bind an epitope containing at least five continuous ordiscontinuous residues from positions 110-147 of SEQ ID NO: 366 withinTNFR2 (CTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGF, SEQ ID NO: 373), or anepitope that exhibits at least 85% sequence identity (e.g., 85%, 90%,95%, 97%, 99%, %, or 100% sequence identity) to this sequence orcontains conservative amino acid substitutions relative to thissequence. For example, in certain cases, agonistic TNFR2 antibodies ofthe invention may specifically bind an epitope that includes one or moreresidues from positions 115-142 of SEQ ID NO: 366 within TNFR2(NRICTCRPGWYCALSKQEGCRLCAPLRK, SEQ ID NO: 369), as well as epitopes thatexhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%,%, or 100% sequence identity) to this sequence and epitopes that containconservative amino acid substitutions relative to this sequence.

Agonistic TNFR2 antibodies of the invention may also specifically bindan epitope containing at least five continuous or discontinuous residuesfrom positions 106-155 of SEQ ID NO: 366 within TNFR2(ETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTE, SEQ ID NO: 374), oran epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%,95%, 97%, 99%, or 100% sequence identity) to this sequence or containsconservative amino acid substitutions relative to this sequence. Forexample, in certain cases, agonistic TNFR2 antibodies of the inventionmay specifically bind an epitope that includes one or more residues frompositions 111-150 of SEQ ID NO: 366 within TNFR2(TREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA, SEQ ID NO: 370), as well asepitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%,95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopesthat contain conservative amino acid substitutions relative to thissequence.

In certain cases, an agonistic TNFR2 antibody of the invention may bindan epitope that includes one or more residues from positions 122-136 ofSEQ ID NO: 366 within TNFR2 (PGWYCALSKQEGCRL, SEQ ID NO: 11), as well asepitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%,95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopesthat contain conservative amino acid substitutions relative to thissequence.

In addition to binding epitopes within TNFR2 that contain one or moreresidues of the KCSPG motif, agonistic TNFR2 antibodies of the inventionmay also bind epitopes containing one or more residues of the KCRPGmotif (positions 142-146 within TNFR2, SEQ ID NO: 375). Preferably,however, agonistic TNFR2 antibodies or antigen-binding fragments thereofbind an epitope containing the KCRPG sequence with a K_(D) that issubstantially higher (e.g., at least 10-fold higher, 15-fold higher,20-fold higher, 25-fold higher, 50-fold higher, 100-fold higher,200-fold higher, 300-fold higher, 400-fold higher, 500-fold higher,600-fold higher, 700-fold higher, 800-fold higher, 900-fold higher,1,000-fold higher, or 10,000-fold higher) than that of the same antibodyor antigen-binding fragment thereof for an epitope containing the KCSPGsequence. For instance, agonistic TNFR2 antibodies of the invention maybind an epitope containing at least five continuous or discontinuousresidues from positions 130-149 of SEQ ID NO: 366 (KQEGCRLCAPLRKCRPGFGV,SEQ ID NO: 357), or an epitope that exhibits at least 85% sequenceidentity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) tothis sequence or contains conservative amino acid substitutions relativeto this sequence. In certain cases, agonistic TNFR2 antibodies of theinvention may bind an epitope containing one or more residues frompositions 137-144 of SEQ ID NO: 366 (CAPLRKCR, SEQ ID NO: 376) and/or anepitope containing one or more residues from positions 141-149 of SEQ IDNO: 366 (KCRPGFGV; SEQ ID NO: 377), as well as epitopes that exhibit atleast 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100%sequence identity) to these sequences and epitopes that containconservative amino acid substitutions relative to these sequences. Inpreferred embodiments, agonistic TNFR2 antibodies or antigen-bindingfragments thereof do not exhibit specific binding for an epitopecontaining the KCRPG sequence.

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention also include those that can discriminate among members of theTNFR superfamily and specifically bind TNFR2 but do not specificallybind other receptors within this family. For instance, in variousembodiments, the invention provides agonistic TNFR2 antibodies that arecapable of binding and activating TNFR2 while not exhibiting specificbinding for a TNFR superfamily member, such as TNFR1, RANK, CD30, CD40,Lymphotoxin beta receptor (LT-PR), OX40, Fas receptor, Decoy receptor 3,CD27, 4-1BB, Death receptor 4, Death receptor 5, Decoy receptor 1, Decoyreceptor 2, Osteoprotegrin, TWEAK receptor, TACI, BAFF receptor,Herpesvirus entry mediator, Nerve growth factor receptor, B-cellmaturation antigen, Glucocorticoid-induced TNFR-related, TROY, Deathreceptor 6, Death receptor 3, and Ectodysplasin A2 receptor.

Agonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention include those that are capable of specifically binding anepitope within human TNFR2 containing one or more, or all, or residues48-67 of SEQ ID NO: 366 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 346), and donot bind any other epitope within TNFR2 or within another TNFRsuperfamily member. For instance, agonistic TNFR2 antibodies orantigen-binding fragments thereof of the invention may specifically bindan epitope within human TNFR2 containing residues 56-60 of SEQ ID NO:366 (KCSPG, SEQ ID NO: 367) and may not specifically bind any otherepitope within TNFR2 or another TNFR superfamily member (e.g., anepitope containing residues 142-146 of human TNFR2 (KCRPG, SEQ ID NO:375), or an epitope containing the residues KCPPG (SEQ ID NO: 396) ofanother TNFR superfamily member).

Agonistic TNFR2 Antibody MR2-1

One example of an agonistic TNFR2 antibody is MR2-1, which binds TNFR2and potentiates TNFR2-mediated T-reg cell proliferation (FIG. 6A). MR2-1binds osteoprotegrin, and is not an antibody of the present invention.However, the heavy and/or light chain variable regions of this antibody,or specifically the heavy and/or light chain CDRs of MR2-1, can bemodified so as to eliminate the capacity of the resulting antibody orfragment thereof to bind a TNFR superfamily member other than TNFR2 soas to produce an agonistic TNFR2 antibody or antigen-binding fragmentthereof of the invention. This can be accomplished using, e.g., geneticengineering and/or antibody library screening techniques describedherein (see, e.g., “Negative screens of antibodies of antigen-bindingfragments” below).

Agonistic TNFR2 Antibody 8E6.D1

A representative agonistic TNFR2 antibody of the invention is 8E6.D1,which is a murine antibody that binds TNFR2 and potentiatesTNFR2-mediated T-reg cell proliferation (FIGS. 6A and 6B). The heavy andlight chain CDRs of 8E6.D1, as well as the heavy and light chainvariable regions in their entirety, and variants of these regions thatexhibit substantially similar specific binding properties of 8E6.D1, canbe used to make an agonistic TNFR2 antibody or antigen-binding fragmentthereof of the invention, e.g., by replacing the mouse constant regionof 8E6.D1 with a non-native constant region (e.g., a constant regionfrom a human antibody).

Agonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention exhibit an affinity for TNFR2 that is the same as or similarto that of 8E6.D1. The high affinity of 8E6.D1 for TNFR2 coupled withthe rapid formation and the slow dissociation of the 8E6.D1-TNFR2complex is consistent with the strong intermolecular contacts thatunderlie this protein-protein interaction. 8E6.D1 binds to distinctepitopes within the primary structure of TNFR2 that are spatiallyaligned in the native conformation of the receptor. The KCSPG motif (SEQID NO: 367) has been identified as a particularly important functionalepitope that establishes strong intermolecular contacts with 8E6.D1 asdetermined by epitope mapping analysis (FIG. 1B). The interaction ofthese residues with TNFR2 antibodies of the invention selectivelypromotes agonistic activity. Agonistic TNFR2 antibody 8E6.D1 does notspecifically bind an epitope containing residues 142-146 within TNFR2(KCRPG, SEQ ID NO: 375). Additionally, 8E6.D1 does not specifically bindany TNFR superfamily member other than TNFR2. 8E6.D1 is capable ofinducing T-reg cell proliferation in a dose-dependent fashion, and isalso capable of synergizing with the cognate ligand for TNFR2, TNF, inorder to promote receptor-mediated T-reg induction (FIGS. 6A and 6B).

Humanized, Primatized, and Chimeric Antibodies

Antibodies of the invention include human, humanized, primatized, andchimeric antibodies that contain one or more of the CDRs of 8E6.D1, or aCDR that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%,99%, or 100% sequence identity) to any of these CDRs or sequences thatcontain conservative mutations relative to these CDRs. Antibodies of theinvention also include human, humanized, primatized, and chimericantibodies that contain one or more CDRs that are identical to those of8E6.D1 except for conservative amino acid substitutions. For example,agonistic TNFR2 antibodies of the invention can be generated byincorporating any of the CDRs of 8E6.D1 into the framework regions(e.g., FR1, FR2, FR3, and FR4) of a human antibody. Exemplary frameworkregions that can be used for the development of a humanized TNFR2antibody containing one or more of the CDRs of 8E6.D1 include, withoutlimitation, those described in U.S. Pat. Nos. 7,732,578, 8,093,068, andWO 2003/105782; the disclosures of which are incorporated herein byreference.

One strategy that can be used to design humanized antibodies of theinvention is to align the sequences of the heavy chain variable regionand light chain variable region of 8E6.D1 with the heavy chain variableregion and light chain variable region of a consensus human antibody.Consensus human antibody heavy chain and light chain sequences are knownin the art (see e.g., the “VBASE” human germline sequence database; seealso Kabat, et al., Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242 (1991); Tomlinson et al., J. Mol. Biol.,227:776-98 (1992); and Cox et al, Eur. J. Immunol., 24:827-836 (1994);the disclosures of each of which are incorporated herein by reference).In this way, the variable domain framework residues and CDRs can beidentified by sequence alignment (see Kabat, supra). One can substituteone or more CDRs of the heavy chain and/or light chain variable domainsof a consensus human antibody with one or more corresponding CDRs of anagonistic TNFR2 antibody of the invention in order to produce ahumanized TNFR2 antibody. Exemplary variable domains of a consensushuman antibody include the heavy chain variable domainEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO: 378)and the light chain variable domainDIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 379), identified inU.S. Pat. No. 6,054,297; the disclosure of which is incorporated hereinby reference (CDRs are shown in bold were determined according to themethod of Chothia, et al., J. Mol. Biol., 196:901-917 (1987), thedisclosure of which is incorporated herein by reference). These aminoacid substitutions can be made, for example, by recombinant expressionof polynucleotides encoding the heavy and light chains of a humanizedantibody in a host cell using methods known in the art or describedherein. For instance, the heavy chain and light chain CDRs of 8E6.D1 canbe inserted into the consensus human antibody heavy and light chainvariable domain sequences in place of the CDRs native to these sequences(shown in bold above) in order to produce a humanized agonistic TNFR2antibody of the invention.

Similarly, this strategy can also be used to produce primatized TNFR2antibodies, as one can substitute the CDRs of the heavy and/or lightchain variable domains of a primate antibody consensus sequence with oneor more corresponding CDRs of 8E6.D1. Consensus primate antibodysequences known in the art (see e.g., U.S. Pat. Nos. 5,658,570;5,681,722; and 5,693,780; the disclosures of each of which areincorporated herein by reference).

In certain cases, it may be desirable to import particular frameworkresidues in addition to CDR sequences from a TNFR2 antibody, such as8E6.D1, into the heavy and/or light chain variable domains of a humanantibody. For instance, U.S. Pat. No. 6,054,297 identifies severalinstances when it may be advantageous to retain certain frameworkresidues from a particular antibody heavy chain or light chain variableregion in the resulting humanized antibody. In certain cases, frameworkresidues may engage in non-covalent interactions with the antigen andthus contribute to the affinity of the antibody for the target antigen.In other cases, individual framework residues may modulate theconformation of a CDR, and thus indirectly influence the interaction ofthe antibody with the antigen. Alternatively, certain framework residuesmay form the interface between V_(H) and V_(L) domains, and maytherefore contribute to the global antibody structure. In other cases,framework residues may constitute functional glycosylation sites (e.g.,Asn-X-Ser/Thr) which may dictate antibody structure and antigen affinityupon attachment to carbohydrate moieties. In cases such as thosedescribed above, it may be beneficial to retain certain frameworkresidues of an agonistic TNFR2 antibody (e.g., 8E6.D1) in the resultinghumanized or primatized antibodies and antigen-binding fragments thereofof the invention, as various framework residues may promote high epitopeaffinity and improved biochemical activity of the antibody orantigen-binding fragment thereof.

Antibodies of the invention also include antibody fragments, Fabdomains, F(ab′)2 molecules, single chain variable fragments (scFvs),tandem scFv fragments, diabodies, triabodies, dual variable domainimmunoglobulins, multi-specific antibodies, bispecific antibodies, SMIPproteins, and heterospecific antibodies that contain one or more of theCDRs of 8E6.D1, or a CDR that exhibits at least 85% sequence identity(e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of theseCDRs. Antibodies and antigen-binding fragments thereof of the inventioninclude those that also contain CDRs having between one and three aminoacid substitutions (e.g., conservative or nonconservative substitutions)relative to the CDR sequences of 8E6.D1. These molecules can beexpressed recombinantly, e.g., by incorporating polynucleotides encodingthese proteins into expression vectors for transfection in a eukaryoticor prokaryotic cell using techniques described herein or known in theart, or synthesized chemically, e.g., by solid phase peptide synthesismethods described herein or known in the art.

Antibodies of the invention additionally include antibody-like scaffoldsthat contain one or more of the CDRs of 8E6.D1, or a CDR that exhibitsat least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100%sequence identity) to any of these CDRs or sequences that containbetween one and three amino acid substitutions (e.g., conservative ornonconservative substitutions) relative to the CDR sequences of 8E6.D1.Examples of antibody-like scaffolds include proteins that contain atenth fibronectin type III domain (¹⁰Fn3), which contains BC, DE, and FGstructural loops analogous to canonical antibodies. It has been shownthat the tertiary structure of the ¹⁰Fn3 domain resembles that of thevariable region of the IgG heavy chain, and one of skill in the art cangraft, e.g., the CDRs of 8E6.D1 or sequences containing conserved aminoacid substitutions relative to these CDRs onto the fibronectin scaffoldby replacing residues of the BC, DE, and FG loops of ¹⁰Fn3 with residuesof 8E6.D1 CDRs. This can be achieved by recombinant expression of amodified ¹⁰Fn3 domain in a prokaryotic or eukaryotic cell (e.g., usingthe vectors and techniques described herein). Examples of using the¹⁰Fn3 domain as an antibody-like scaffold for the grafting of CDRs fromantibodies onto the BC, DE, and FG structural loops are reported in WO2000/034784; WO 2009/142773; WO 2012/088006; and U.S. Pat. No.8,278,419; the disclosures of each of which are incorporated herein byreference.

Nucleic Acids and Expression Systems

Agonistic TNFR2 antibodies of the invention can be prepared by any of avariety of established techniques. For instance, an agonistic TNFR2antibody or antigen-binding fragment thereof of the invention can beprepared by recombinant expression of immunoglobulin light and heavychain genes in a host cell. To express an antibody recombinantly, a hostcell can be transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and heavychains of the antibody such that the light and heavy chains areexpressed in the host cell and, optionally, secreted into the medium inwhich the host cells are cultured, from which medium the antibodies canbe recovered. Standard recombinant DNA methodologies are used to obtainantibody heavy and light chain genes, incorporate these genes intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Molecular Cloning; A LaboratoryManual, Second Edition (Sambrook, Fritsch and Maniatis (eds), ColdSpring Harbor, N. Y., 1989), Current Protocols in Molecular Biology(Ausubel et al., eds., Greene Publishing Associates, 1989), and in U.S.Pat. No. 4,816,397; the disclosures of each of which are incorporatedherein by reference.

Vectors for Expression of Agonistic TNFR2 Antibodies and AntibodyFragments

Viral genomes provide a rich source of vectors that can be used for theefficient delivery of exogenous genes into the genome of a cell (e.g., aeukaryotic or prokaryotic cell). Viral genomes are particularly usefulvectors for gene delivery because the polynucleotides contained withinsuch genomes are typically incorporated into the genome of a target cellby generalized or specialized transduction. These processes occur aspart of the natural viral replication cycle, and do not require addedproteins or reagents in order to induce gene integration. Examples ofviral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34,Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses),coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g.,influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitisvirus), paramyxovirus (e.g. measles and Sendai), positive strand RNAviruses, such as picornavirus and alphavirus, and double stranded DNAviruses including adenovirus, herpes virus (e.g., Herpes Simplex virustypes 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g.,vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Otherviruses useful for delivering polynucleotides encoding antibody lightand heavy chains or antibody fragments of the invention include Norwalkvirus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, andhepatitis virus, for example. Examples of retroviruses include: avianleukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses,HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: Theviruses and their replication, In Fundamental Virology, Third Edition,B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia,1996, the disclosure of which is incorporated herein by reference).Other examples of viral genomes useful in conjunction with thecompositions and methods of the invention include murine leukemiaviruses, murine sarcoma viruses, mouse mammary tumor virus, bovineleukemia virus, feline leukemia virus, feline sarcoma virus, avianleukemia virus, human T-cell leukemia virus, baboon endogenous virus,Gibbon ape leukemia virus, Mason Pfizer monkey virus, simianimmunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, andlentiviruses. Other examples of vectors are described, for example, inU.S. Pat. No. 5,801,030; the disclosure of which is incorporated hereinby reference.

Genome Editing Techniques

In addition to viral vectors, a variety of additional methods have beendeveloped for the incorporation of genes, e.g., those encoding antibodylight and heavy chains, single chain variable fragments (scFvs), tandemscFvs, Fab domains, F(ab′)₂ domains, diabodies, and triabodies, amongothers, into the genomes of target cells for antibody expression. Onesuch method that can be used for incorporating polynucleotides encodingagonistic TNFR2 antibodies or antigen-binding fragments thereof intoprokaryotic or eukaryotic cells includes transposons. Transposons arepolynucleotides that encode transposase enzymes and contain apolynucleotide sequence or gene of interest flanked by excision sites atthe 5′ and 3′ positions. Once a transposon has been delivered into acell, expression of the transposase gene commences and results in activeenzymes that cleave the gene of interest from the transposon. Thisactivity is mediated by the site-specific recognition of transposonexcision sites by the transposase. In certain cases, these excisionsites may be terminal repeats or inverted terminal repeats. Once excisedfrom the transposon, the gene of interest can be integrated into thegenome of a prokaryotic or eukaryotic cell by transposase-catalyzedcleavage of similar excision sites that exist within nuclear genome ofthe cell. This allows the gene encoding a TNFR2 antibody or fragment ordomain thereof to be inserted into the cleaved nuclear DNA at theexcision sites, and subsequent ligation of the phosphodiester bonds thatjoin the gene of interest to the DNA of the prokaryotic or eukaryoticcell genome completes the incorporation process. In certain cases, thetransposon may be a retrotransposon, such that the gene encoding theantibody is first transcribed to an RNA product and thenreverse-transcribed to DNA before incorporation in the prokaryotic oreukaryotic cell genome. Exemplary transposon systems include thepiggybac transposon (described in detail in WO 2010/085699) and thesleeping beauty transposon (described in detail in US20050112764); thedisclosures of each of which are incorporated herein by reference.

Another useful method for the integration of nucleic acid moleculesencoding agonistic TNFR2 antibodies or antigen-binding fragments thereofinto the genome of a prokaryotic or eukaryotic cell utilizes clusteredregularly interspaced short palindromic repeats (CRISPR)/Cas technology,which is derived from a system that originally evolved as an adaptivedefense mechanism in bacteria and archaea against infection by viruses.The CRISPR/Cas system consists of palindromic repeat sequences withinplasmid DNA and an associated Cas9 nuclease. This ensemble of DNA andprotein directs site specific DNA cleavage of a target sequence by firstincorporating foreign DNA into CRISPR loci. Polynucleotides containingthese foreign sequences and the repeat-spacer elements of the CRISPRlocus are in turn transcribed in a host cell to create a guide RNA,which can subsequently anneal to a target sequence and localize the Cas9nuclease to this site. In this manner, highly site-specificcas9-mediated DNA cleavage can be engendered in a foreign polynucleotidebecause the interaction that brings cas9 within close proximity of thetarget DNA molecule is governed by RNA:DNA hybridization. As a result,one can theoretically design a CRISPR/Cas system to cleave any targetDNA molecule of interest. This technique has been exploited in order toedit eukaryotic genomes (Hwang et al., Nat. Biotech., 31:227-229 (2013),the disclosure of which is incorporated herein by reference) and can beused as an efficient means of site-specifically editing eukaryotic orprokaryotic genomes in order to cleave DNA prior to the incorporation ofa polynucleotide encoding a TNFR2 antibody or antigen-binding fragmentthereof of the invention. The use of CRISPR/Cas to modulate geneexpression has been described in U.S. Pat. No. 8,697,359; the disclosureof which is incorporated herein by reference.

Alternative methods for site-specifically cleaving genomic DNA prior tothe incorporation of a polynucleotide encoding a TNFR2 antibody orantibody fragment of the invention include the use of zinc fingernucleases and transcription activator-like effector nucleases (TALENs).Unlike the CRISPR/Cas system, these enzymes do not contain a guidingpolynucleotide to localize to a specific target sequence. Targetspecificity is instead controlled by DNA binding domains within theseenzymes. Zinc finger nucleases and TALENs for use in genome editingapplications are described, e.g., in Urnov et al., Nat. Rev. Genet.,11:636-646 (2010); and in Joung et al., Nat. Rev. Mol. Cell. Bio.,14:49-55 (2013); the disclosures of each of which are incorporatedherein by reference. Additional genome editing techniques that can beused to incorporate polynucleotides encoding antibodies of the inventioninto the genome of a prokaryotic or eukaryotic cell include the use ofARCUS™ meganucleases that can be rationally designed so as tosite-specifically cleave genomic DNA. The use of these enzymes for theincorporation of polynucleotides encoding agonistic TNFR2 antibodies orantibody fragments of the invention into the genome of a prokaryotic oreukaryotic cell is particularly advantageous in view of thestructure-activity relationships that have been established for theseenzymes. Single chain meganucleases can thus be modified at certainamino acid positions in order to create nucleases that selectivelycleave DNA at desired locations. These single-chain nucleases have beendescribed extensively, e.g., in U.S. Pat. Nos. 8,021,867 and 8,445,251;the disclosures of each of which are incorporated herein by reference.

Polynucleotide Sequence Elements

To express agonistic TNFR2 antibodies or antibody fragments of theinvention, polynucleotides encoding partial or full-length light andheavy chains, e.g., obtained as described above, can be inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used. Polynucleotides encoding the light chaingene and the heavy chain of a TNFR2 antibody can be inserted intoseparate vectors, or, optionally, both polynucleotides can beincorporated into the same expression vector using establishedtechniques described herein or known in the art.

In addition to polynucleotides encoding the heavy and light chains of anantibody (or a polynucleotide encoding an antibody fragment, such as ascFv molecule), the recombinant expression vectors of the invention maycarry regulatory sequences that control the expression of the antibodychain genes in a host cell. The design of the expression vector,including the selection of regulatory sequences, may depend on suchfactors as the choice of the host cell to be transformed or the level ofexpression of protein desired. For instance, suitable regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)) and polyoma. For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. Nos.5,168,062; 4,510,245; and 4,968,615; the disclosures of each of whichare incorporated herein by reference.

In addition to antibody heavy and light chain genes and regulatorysequences, recombinant expression vectors of the invention may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. A selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216; 4,634,665; and 5,179,017; the disclosures of each ofwhich are incorporated herein by reference). For example, typically theselectable marker gene confers resistance to cytotoxic drugs, such asG418, puromycin, blasticidin, hygromycin or methotrexate, to a host cellinto which the vector has been introduced. Suitable selectable markergenes include the dihydrofolate reductase (DHFR) gene (for use in DHFR″host cells with methotrexate selection/amplification) and the neo gene(for G418 selection). In order to express the light and heavy chains ofa TNFR2 antibody or a TNFR2 antibody fragment, the expression vector(s)containing polynucleotides encoding the heavy and light chains can betransfected into a host cell by standard techniques known in the art ordescribed herein.

Polynucleotides Encoding Modified Agonistic TNFR2 Antibodies andAntibody Fragments

In certain cases, agonistic TNFR2 antibodies or antibody fragments ofthe invention can be produced that are similar to a particular agonisticTNFR2 antibody but feature differences in the sequence of one or moreCDRs. In other cases, the antibodies of the invention may be similarfeature differences in one or more framework regions relative to anotheragonistic TNFR2 antibody. For instance, one or more framework regions ofan agonistic TNFR2 antibody derived from a non-human mammal may besubstituted with the framework region of a human antibody. Exemplaryframework regions include, for example, human framework regionsdescribed in U.S. Pat. No. 7,829,086, and primate framework regions asdescribed in EP 1945668; the disclosures of each of which areincorporated herein by reference. Alternatively, antibodies of theinvention may be similar to another agonistic TNFR2 antibody but exhibitdifferences in the sequence of one or more CDRs and differences in oneor more framework regions. To generate nucleic acids encoding suchagonistic TNFR2 antibodies, DNA fragments encoding, e.g., at least one,or both, of the light chain variable regions and the heavy chainvariable regions can be produced by chemical synthesis (e.g., by solidphase polynucleotide synthesis techniques), in vitro gene amplification(e.g., by polymerase chain reaction techniques), or by replication ofthe polynucleotide in a host organism. For instance, nucleic acidsencoding agonistic TNFR2 antibodies of the invention may be obtained byamplification and modification of germline DNA or cDNA encoding lightand heavy chain variable sequences so as to incorporate the CDRs of anagonistic TNFR2 antibody into the framework residues of a consensusantibody. This can be achieved, for example, by performing site-directedmutagenesis of germline DNA or cDNA and amplifying the resultingpolynucleotides using the polymerase chain reaction (PCR) according toestablished procedures. Germline DNA sequences for human heavy and lightchain variable region genes are known in the art (see, e.g., the “VBASE”human germline sequence database; see also Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242 (1991); Tomlinsonet al., J. Mol. Biol., 227:776-798 (1992); and Cox et al., Eur. J.Immunol., 24:827-836 (1994); the disclosures of each of which areincorporated herein by reference). Additionally, a polynucleotideencoding the heavy or light chain variable region of an agonistic TNFR2antibody can be synthesized and used as a template for mutagenesis togenerate a variant as described herein using routine mutagenesistechniques. Alternatively, a DNA fragment encoding the variant can bedirectly synthesized (e.g., by established solid phase nucleic acidchemical synthesis procedures).

The isolated DNA encoding the V_(H) region of an agonistic TNFR2antibody of the invention can be converted to a full-length heavy chaingene (as well as a Fab heavy chain gene) by operatively linking theV_(H)-encoding DNA to another DNA molecule encoding heavy chain constantregion domains (CH1, CH2, CH3, and, optionally, CH4). The sequences ofhuman heavy chain constant region genes are known in the art (see e.g.,Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242, 1991, the disclosure of which is incorporated herein byreference) and DNA fragments encompassing these regions can be obtainedby standard PCR amplification. The heavy chain constant region can be anIgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but incertain embodiments is an IgG1 constant region. For a Fab fragment heavychain gene, the VH-encoding DNA can be operatively linked to another DNAmolecule encoding only the heavy chain CH1 domain.

The isolated DNA encoding the VL region of an agonistic TNFR2 antibodyof the invention can be converted to a full-length light chain gene (aswell as a Fab light chain gene) by operatively linking the VL-encodingDNA to another DNA molecule encoding the light chain constant region,CL. The sequences of human light chain constant region genes are knownin the art (see e.g., Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition (U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, 1991)) and DNA fragmentsencompassing these regions can be obtained, e.g., by amplification in aprokaryotic or eukaryotic cell of a polynucleotide encoding theseregions, by PCR amplification, or by chemical polynucleotide synthesis.The light chain constant region can be a kappa (κ) or lambda (A)constant region. To create a scFv gene, the VH and VL-encoding DNAfragments are operatively linked to another fragment encoding a flexiblelinker, e.g., a polynucleotide encoding a flexible, hydrophilic aminoacid sequence, such as the amino acid sequence (Gly₄Ser)₃, such that theVH and VL sequences can be expressed as a contiguous single-chainprotein, with the VL and VH regions joined by the linker (see e.g., Birdet al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad.Sci. USA 85:5879-5883 (1988); McCafferty et al., Nature 348:552-554(1990); the disclosures of each of which are incorporated herein byreference).

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to TNFR2. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies can be produced in whichone heavy and one light chain are derived from an agonistic TNFR2antibody and the other heavy and light chain are specific for an antigenother than TNFR2. Such antibodies can be generated, e.g., bycrosslinking a heavy chain and light chain derived from an agonisticTNFR2 antibody to a heavy chain and light chain of a second antibody bystandard chemical crosslinking methods (e.g., by disulfide bondformation). Bifunctional antibodies can also be made by expressing anucleic acid molecule engineered to encode a bifunctional antibody in aprokaryotic or eukaryotic cell.

In certain cases, dual specific antibodies, i.e., antibodies that bindTNFR2 and a different antigen using the same binding site, can beproduced by mutating amino acid residues in the light chain and/or heavychain CDRs. In various embodiments, dual specific antibodies that bindtwo antigens, such as TNFR2 and a second cell-surface receptor, can beproduced by mutating amino acid residues in the periphery of theantigen-binding site. (Bostrom et al., Science 323: 1610-1614 (2009);the disclosure of which is incorporated herein by reference). Dualfunctional antibodies can be made by expressing a polynucleotideengineered to encode a dual specific antibody.

Modified agonistic TNFR2 antibodies and antibody fragments of theinvention can also be produced by chemical synthesis (e.g., by themethods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 ThePierce Chemical Co., Rockford, 111; the disclosure of which isincorporated herein by reference). Variant antibodies can also begenerated using a cell-free synthetic platform (see, e.g., Chu et al.,Biochemia No. 2, 2001 (Roche Molecular Biologicals); the disclosure ofwhich is incorporated herein by reference).

The above-described methods can be applied to antibody 8E6.D1 so as toproduce an agonistic TNFR2 antibody or antigen-binding fragment thereofwith altered properties. For instance, agonistic TNFR2 antibodies of theinvention can be based on the heavy chain or light chain amino acidsequences or one or more CDRs of 8E6.D1. Using techniques describedherein or known in the art, one can modify one or more amino acidsequences of 8E6.D1, e.g., so as to improve the affinity of theresulting antibody for TNFR2. Full-length antibodies and antibodyfragments can also be produced using the amino acid sequences of 8E6.D1as a starting point for the design of other TNFR2 agonistic antibodiesor antigen-binding fragments of the invention. For instance, usingstandard techniques known in the art, one can produce DNA fragmentsencoding the VH and/or VL segments of 8E6.D1, e.g., by chemicalsynthesis or by PCR-based methods. These DNA fragments can be furthermanipulated by standard recombinant DNA techniques, e.g., to convert thevariable region genes to full-length antibody chain genes or to fragmentgenes, such as those that encode a Fab fragment, F(ab′)2 fragment, scFv,diabody, triabody, or antibody-like scaffold protein. In thesemanipulations, a VL- or VH-encoding DNA fragment is operatively linkedto another DNA fragment encoding another protein, such as an antibodyconstant region, a flexible linker, or a scaffold protein (e.g., a ¹⁰Fn3domain).

Host Cells for Expression of Agonistic TNFR2 Antibodies and AntibodyFragments

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention may be expressed in either prokaryotic or eukaryotic hostcells. In certain embodiments, expression of antibodies orantigen-binding fragments thereof is performed in eukaryotic cells,e.g., mammalian host cells, for optimal secretion of a properly foldedand immunologically active antibody. Exemplary mammalian host cells forexpressing the recombinant antibodies or antigen-binding fragmentsthereof of the invention include Chinese Hamster Ovary (CHO cells)(including DHFR CHO cells, described in Urlaub and Chasin, Proc. Natl.Acad. Sci. USA, 77:4216-4220 (1980), used with a DHFR selectable marker,e.g., as described in Kaufman and Sharp, Mol. Biol., 159:601-621,(1982), NSO myeloma cells, COS cells, 293 cells, and SP2/0 cells.Additional cell types that may be useful for the expression ofantibodies and fragments thereof include bacterial cells, such asBL-21(DE3) E. coli cells, which can be transformed with vectorscontaining foreign DNA according to established protocols. Additionaleukaryotic cells that may be useful for expression of antibodies includeyeast cells, such as auxotrophic strains of S. cerevisiae, which can betransformed and selectively grown in incomplete media according toestablished procedures known in the art. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or secretion of the antibody into the culture medium in whichthe host cells are grown.

Antibodies can be recovered from the culture medium using standardprotein purification methods. Host cells can also be used to produceportions of intact antibodies, such as Fab fragments or scFv molecules.The invention also includes methods in which the above procedure isvaried according to established protocols known in the art. For example,it can be desirable to transfect a host cell with DNA encoding eitherthe light chain or the heavy chain (but not both) of an agonistic TNFR2antibody of this invention in order to produce an antigen-bindingfragment of the antibody.

Once an agonistic TNFR2 antibody or antigen-binding fragments thereof ofthe invention has been produced by recombinant expression, it can bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for TNFR2 after Protein Aor Protein G selection, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. Further, the agonistic TNFR2antibodies of the invention or fragments thereof can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification or to produce therapeuticconjugates (see “Antibody conjugates,” below).

Once isolated, an agonistic TNFR2 antibody or antigen-binding fragmentsthereof can, if desired, be further purified, e.g., by high performanceliquid chromatography (see, e.g., Fisher, Laboratory Techniques inBiochemistry and Molecular Biology (Work and Burdon, eds., Elsevier,1980); the disclosure of which is incorporated herein by reference), orby gel filtration chromatography, such as on a Superdex™ 75 column(Pharmacia Biotech AB, Uppsala, Sweden).

Platforms for Generating and Affinity-Maturing Agonistic TNFR2Antibodies and Antigen-Binding Fragments

Mapping Epitopes of TNFR2 that Promote Receptor Activation

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention can be produced by screening libraries of antibodies andantigen-binding fragments thereof for functional molecules that arecapable of binding epitopes within TNFR2 that selectively promotereceptor activation. Linear peptides isolated from the TNFR2 protein maynot adopt the same three dimensional conformations as those peptidesequences located within the protein. TNFR2 provides a structurallyrigidified framework that biases the conformations of individual peptidefragments and reinforces these spatial orientations by establishingintramolecular contacts (e.g., hydrogen bonds, dipole-dipoleinteractions, salt bridges) and by differentially positioning variousregions for exposure to solvent depending on the relative hydrophilicityand lipophilicity of these areas (Mukai et al., Sci. Signal.,3:ra83-ra83 (2010); the disclosure of which is incorporated herein byreference). The conformational constraint of a peptide fragment withinTNFR2 can be achieved by incorporating the amino acid residues of aTNFR2 epitope (e.g., an epitope that promotes receptor activation) intoa structurally pre-organized peptide scaffold, such as a cyclic,bicyclic, tricyclic, or tetrayclic peptide. Cyclic and polycyclicpeptides such as these confine a peptide fragment to a distinctthree-dimensional conformation. This can be achieved, e.g., bysynthesizing peptide epitopes isolated from TNFR2 by establishedchemical synthetic methods (e.g., by solid phase peptide synthesis asdescribed herein) and incorporating cysteine residues into the sequenceat the N- and C-terminal positions or at various internal positionswithin the peptide chain. It may be advantageous to incorporate cysteineresidues that are chemically protected at the thiol moiety with aprotecting group that can be removed under conditions different fromthose used to remove other protecting groups within the peptide beingsynthesized and different from those used to assemble the peptide chain.Exemplary orthogonal protecting groups for the cysteine thiol includethe 4-methyltrityl group and 4-methoxtrityl group, each of which can beremoved using dilute trifluoracetic acid (examples are described, e.g.,in Isidro-Llobet et al., Chem Rev., 109:2455-2504 (2009); the disclosureof which is incorporated herein by reference).

After introducing a cysteine residue into a synthetic peptide fragmentderived from an epitope within TNFR2, the peptide can be cyclized bytreating the peptide with a multivalent electrophile, such as abis(bromomethyl) or tris(bromomethyl)arene derivative. Alternativemultivalent thiol-reactive electrophiles can be used, e.g.,1,5-difluoro-2,4-dinitrobenzene, acyclic dibromoalkanes, and others(see, e.g., Jo et al., J. Am. Chem. Soc., 134:17704-17713 (2012); thedisclosure of which is incorporated herein by reference). In certaincases, it may be advantageous to prevent the participation of a cysteineresidue in the synthetic peptide fragment in a cyclization reaction. Forinstance, it may be desirable to synthesize a polycyclic peptidecontaining multiple cysteine residues such that only select cysteinethiols participate in the intramolecular crosslinking process. Toprevent unwanted participation of these additional Cys thiol groups inthe coupling reaction, a simple approach is, for instance, to useFmoc-Cys(Acm) (Fmoc-acetamidomethyl-L-cysteine) for the introduction ofa protected Cys residue during the course of peptide synthesis.Alternatively, Fmoc-Cys(StBu)-OH can be used, and/or the correspondingt-butyloxycarbonyl (Boc)-protected amino acids. The Acm or StBu group isnot removed during the course of a normal TFA deprotection-cleavagereaction, and instead requires oxidative treatment (e.g., with iodine,12) in the case of the Acm group, or reductive treatment (e.g.,β-mercaptoethanol or 1,4-dithiothreiotol) in the case of the StBu groupto yield the reduced sulfhydryl form of the peptide, which can either beused directly or subsequently oxidized to the corresponding cystinylpeptide. In one embodiment, a peptide is used which contains at leastone Cys derivative, such as Cys(Acm) or Cys(StBu), to allow selectivedeprotection of a Cys-thiol group. Selective deprotection of a Cys-thiolgroup renders the Cys-thiol group available for reacting at a desiredmoment, such as following completion of peptide chain assembly and priorto the deprotection of other residues within the peptide (see, e.g., WO2008/013454; the disclosure of which is incorporated herein byreference).

As an example, libraries of cyclic and polycyclic peptides containingindividual fragments isolated from TNFR2 and combinations of fragmentsfrom distinct regions of TNFR2 can be synthesized by techniques such asthose described above in order to incorporate cysteine residues atvarious positions within the peptide scaffold and using differentelectrophilic crosslinking reagents (see, e.g., Example 1 and FIG. 2A,SEQ ID NOs: 1-341). These peptides can be immobilized on a solid surfaceand screened for molecules that bind MR2-1 using an ELISA-basedscreening platform using established procedures. Using this assay,peptides that contain residues within epitopes of TNFR2 that promotereceptor activation may structurally pre-organize these amino acids suchthat they resemble the conformations of the corresponding peptide in thenative protein. Cyclic and polycyclic peptides thus obtained (e.g.,peptides having the sequence of any one of SEQ ID NOs: 1-341, andparticularly those that contain the KCSPG motif, as in SEQ ID NOs: 53,69, 75, 118, and 233) can be used to screen libraries of antibodies andantigen-binding fragments thereof in order to identify TNFR2 antibodiesof the invention. Moreover, since these constrained peptides act assurrogates for epitopes within TNFR2 that promote receptor activation,antibodies generated using this screening technique may bind thecorresponding epitopes in TNFR2 and are expected to be agonistic ofreceptor activity.

Screening of Antibody Libraries for Agonistic TNFR2 Antibodies andAntigen-Binding Fragments

Methods for high throughput screening of antibody libraries formolecules capable of binding epitopes within TNFR2 (e.g., epitopespresented by peptides having the sequence of any one of SEQ ID NOs:1-341, such as SEQ ID NOs: 3, 11, 61, or 87, and particularly those thatcontain the KCSPG motif, as in SEQ ID NOs: 53, 69, 75, 118, and 233)include, without limitation, display techniques including phage display,bacterial display, yeast display, mammalian display, ribosome display,mRNA display, and cDNA display. The use of phage display to isolateligands that bind biologically relevant molecules has been reviewed,e.g., in Felici et al., Biotechnol. Annual Rev. 1:149-183 (1995); Katz,Annual Rev. Biophys. Biomol. Struct. 26:27-45 (1997); and Hoogenboom etal., Immunotechnology 4:1-20, (1998); the disclosures of each of whichare incorporated herein by reference. Several randomized combinatorialpeptide libraries have been constructed to select for polypeptides thatbind different targets, e.g., cell surface receptors or DNA (reviewed byKay, Perspect. Drug Discovery Des. 2, 251-268 (1995); and Kay et al.,Mol. Divers. 1:139-140 (1996). Proteins and multimeric proteins havebeen successfully phage-displayed as functional molecules (see EP0349578A, EP 4527839A, EP 0589877A; Chiswell and McCafferty, TrendsBiotechnol. 10, 80-84 (1992)). In addition, functional antibodyfragments (e.g. Fab, single chain Fv [scFv]) have been expressed (see,e.g., McCafferty et al., Nature 348: 552-554 (1990); Barbas et al.,Proc. Natl. Acad Sci. USA 88:7978-7982 (1991); and Clackson et al.,Nature 352:624-628 (1991). These references are hereby incorporated byreference in their entirety.

Phage Display Techniques

As an example, phage display techniques can be used in order to screenlibraries of antibodies and antigen-binding fragments thereof forfunctional molecules capable of binding cyclic or polycyclic peptidescontaining epitopes within TNFR2 that promote receptor activation (e.g.,peptides having the sequence of any one of SEQ ID NOs: 1-341, such asSEQ ID NOs: 3, 11, 61, or 87, and particularly those that contain theKCSPG motif, as in SEQ ID NOs: 53, 69, 75, 118, and 233). For instance,libraries of polynucleotides encoding single chain antibody fragments,such as scFv fragments, that contain randomized hypervariable regionscan be obtained using established procedures (e.g., solid phasepolynucleotide synthesis or error-prone PCR techniques, see McCullum etal., Meth. Mol. Biol., 634:103-(2010); incorporated herein byreference). These randomized polynucleotides can subsequently beincorporated into a viral genome such that the randomized antibodychains encoded by these genes are expressed on the surface offilamentous phage, e.g., by a covalent bond between the antibody chainand a coat protein (e.g., pill coat protein on the surface of M13phage). This provides a physical connection between the genotype andphenotype of the antibody chain. In this way, libraries of phage thatdisplay diverse antibody chains containing random mutations inhypervariable regions can be screened for the ability of the exteriorantibody chains to bind TNFR2 epitopes (e.g., peptides having thesequence of any one of SEQ ID NOs: 1-341) that are immobilized to asurface using established procedures. For instance, cyclic peptides suchas those represented by SEQ ID NOs: 53, 69, 75, 118, and 233, whichcontain the KCSPG motif, can be physically bound to the surface of amicrotiter plate by forming a covalent bond between the peptide and anepitope tag (e.g., biotin) and incubating the peptide in wells of amicrotiter plate that have been previously coated with a complementarytag (e.g., avidin) that binds the tag attached to the peptide with highaffinity. Suitable epitope tags include, without limitation,maltose-binding protein, glutathione-S-transferase, a poly-histidinetag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag,biotin, streptavidin. Peptides containing the epitopes presented bythese molecules are capable of being immobilized on surfaces containingsuch complementary molecules as maltose, glutathione, anickel-containing complex, an anti-FLAG antibody, an anti-myc antibody,an anti-HA antibody, streptavidin, or biotin, respectively. In this way,phage can be incubated with a surface containing an immobilizedTNFR2-derived peptide for a time suitable to allow binding of theantibody to the constrained peptide and in the presence of anappropriate buffer system (e.g., one that contains physiological saltconcentration, ionic strength, and is maintained at physiological pH bya buffering agent). The surface can then be washed (e.g., with phosphatebuffer containing 0.1% Tween-20) so as to remove phage that do notpresent antibody chains that interact with the TNFR2-derived peptideswith an affinity greater than a particular threshold value.

The affinity of the antibodies that remain after this initial panning(i.e., screening) step can be modulated by adjusting the conditions ofthe washing step (e.g., by including mildly acidic or basic components,or by including other TNFR2-derived peptides at a low concentration inorder to compete with immobilized peptides for antigen-binding sites).In this way, the population of phage that remains bound to the surfacesof the microtiter plate following the washing step is enriched for phagethat bind TNFR2-derived peptide epitopes that promote receptoractivation. The remaining phage can then be amplified by eluting thephage from the surface containing these peptides (e.g., by altering theambient pH, ionic strength, or temperature) so as to diminishprotein-protein interaction strength. The isolated phage can then beamplified, e.g., by infecting bacterial cells, and the resulting phagecan optionally be subjected to panning by additional iterations ofscreening so as to further enrich the population of phage for thoseharboring higher-affinity TNFR2 antibodies. Following these panningstages, phage that display high-affinity antibodies or antigen-bindingfragments thereof can subsequently be isolated and the genomes of thesephage can be sequenced in order to identify the polynucleotide andpolypeptide sequences of the encoded antibodies. Phage displaytechniques such as this can be used to generate, e.g., antibody chains,such as scFv fragments, tandem scFv fragments, and other antigen-bindingfragments of the invention that can be used as agonists of TNFR2.Exemplary phage display protocols for the identification of antibodychains and antigen-binding fragments thereof that bind a particularantigen with high affinity are well-established and are described, e.g.,in U.S. Pat. No. 7,846,892; WO 1997/002342; U.S. Pat. No. 8,846,867; andWO 2007/132917; the disclosures of which are incorporated herein byreference. Similar phage display techniques can be used to generateantibody-like scaffolds (e.g., ¹⁰Fn3 domains) of the invention that bindepitopes within TNFR2 that promote receptor activation (e.g., epitopespresented by peptides with the sequence of any one of SEQ ID NOs: 1-341,such as SEQ ID NOs: 3, 11, 61, or 87, and particularly those thatcontain the KCSPG motif, as in SEQ ID NOs: 53, 69, 75, 118, and 233).Exemplary phage display protocols for the identification ofantibody-like scaffold proteins are described, e.g., in WO 2009/086116;the disclosure of which is incorporated herein by reference).

(ii) Cell-Based Display Techniques

Other in vitro display techniques that exploit the linkage betweengenotype and phenotype of a solvent-exposed antibody or antigen-bindingfragment thereof include yeast and bacterial display. Yeast displaytechniques are established in the art and are often advantageous in thathigh quantities of antibodies (often up to 30,000) can be presented onthe surface of an individual yeast cell (see, e.g., Boder et al., NatBiotechnol. 15:553 (1997); the disclosure of which is incorporatedherein by reference). The larger size of whole cells (e.g., yeast cellsor bacterial cells) over filamentous phage enables an additionalscreening strategy, as one can use flow cytometry to both analyze andsort libraries of labeled cells. For instance, established procedurescan be used to generate libraries of bacterial cells or yeast cells thatexpress antibodies containing randomized hypervariable regions (see,e.g., see U.S. Pat. No. 7,749,501 and US 2013/0085072; the disclosuresof each which are incorporated herein by reference). For instance, largelibraries of yeast cells that express polynucleotides encoding naïvescFv fragments can be made using established procedures (de Bruin etal., Nat Biotechnol 17:397, (1999); the disclosure of which isincorporated herein by reference). Yeast cells expressing thesepolynucleotides can then be incubated with two different fluorescentmolecules during the panning steps: one dye that binds conservedresidues within the antibody and thus reflects the amount of antibodydisplayed, and another dye that fluoresces at a different wavelength andbinds the antigen, thus indicating the amount of antigen bound. In thesecases, it is useful to use a cyclic or polycyclic peptide containing thesequence of any one of SEQ ID NOs: 1-341, such as SEQ ID NOs: 3, 11, 61,or 87 (and particularly those that contain the KCSPG motif, as in SEQ IDNOs: 53, 69, 75, 118, and 233) that has been conjugated to an epitopetag (e.g., biotin), optionally at a residue that is not expected tointerfere with antibody-antigen-binding. This enables a fluorescent dyelabeled with a complementary tag (e.g., avidin) to localize to theantibody-antigen complex. This results in great flexibility andimmediate feedback on the progress of a selection. In contrast to phagedisplay, by normalizing to antibody display levels, antibodies withhigher affinities, rather than greater expression levels can easily beselected. In fact, it is possible to distinguish and sort antibodieswhose affinities differ by only two-fold (see, e.g., VanAntwerp andWittrup, Biotechnol. Prog., 16:31, (2000); the disclosure of which isincorporated herein by reference).

(iii) Nucleotide Display Techniques

Display techniques that utilize in vitro translation of randomizedpolynucleotide libraries also provide a powerful approach to generatingagonistic TNFR2 antibodies of the invention. For instance, randomizedDNA libraries encoding antibodies or antigen-binding fragments thereofthat contain mutations within designated hypervariable regions can beobtained, e.g., using established PCR-based mutagenesis techniques asdescribed herein. The polynucleotides of these libraries may containtranscription regulating sequences, such as promoters and transcriptionterminating sequences, and may additionally encode sequences thatincrease the rate of translation of the resulting mRNA construct (e.g.,RES sequences, 5′ and 3′ UTRs, a poly-adenylation tract, and otherelements known in the art to promote translation of an RNA transcript).These polynucleotide libraries can be incubated in an appropriatelybuffered solution containing RNA polymerase and RNA nucleosidetriphosphates (NTPs) in order to enable transcription of the DNAsequences to competent mRNA molecules, which can subsequently betranslated by large and small ribosomal subunits, aminoacyl tRNAmolecules, and translation initiation and elongation factors present insolution (e.g., using the PURExpress® In Vitro Protein Synthesis Kit,New England Biolabs®). Designed mRNA modifications can enable theantibody product to remain covalently bound to the mRNA template by achemical bond to puromycin (e.g., see Keefe, Curr. Protoc. Mol. Biol.,Chapter 24, Unit 24.5 (2001); the disclosure of which is incorporatedherein by reference). This genotype-phenotype linkage can thus be usedto select for antibodies that bind a TNFR2-derived peptide (e.g., apeptide that has the sequence of any one of SEQ ID NOs: 1-341, such asSEQ ID NOs: 3, 11, 61, or 87, and particularly those that contain theKCRPG motif, as in SEQ ID NOs: 53, 69, 75, 118, and 233) by incubatingmRNA:antibody fusion constructs with a peptide immobilized to a surfaceand panning in a fashion similar to phage display techniques (see, e.g.,WO 2006/072773; the disclosure of which is incorporated herein byreference).

Optionally, antibodies of the invention can be generated using a similartechnique, except the antibody product may be bound non-covalently tothe ribosome-mRNA complex rather than covalently via a puromycin linker.This platform, known as ribosome display, has been described, e.g., inU.S. Pat. No. 7,074,557; the disclosure of which is incorporated hereinby reference. Alternatively, antibodies can be generated using cDNAdisplay, a technique that is analogous to mRNA display methodology withthe exception that cDNA, rather than mRNA, is covalently bound to anantibody product via a puromycin linker. cDNA display techniques offerthe advantage of being able to perform panning steps under increasinglystringent conditions, e.g., under conditions in which the saltconcentration, ionic strength, pH, and/or temperature of the environmentis adjusted in order to screen for antibodies with particularly highaffinity for TNFR2-derived peptides. This is due to the higher naturalstability of double-stranded cDNA over single-stranded mRNA. cDNAdisplay screening techniques are described, e.g., in Ueno et al.,Methods Mol. Biol., 805:113-135 (2012); the disclosure of which isincorporated herein by reference.

In addition to generating agonistic TNFR2 antibodies of the invention,in vitro display techniques (e.g., those described herein and thoseknown in the art) also provide methods for improving the affinity of aTNFR2 antibody of the invention. For instance, rather than screeninglibraries of antibodies and fragments thereof containing completelyrandomized hypervariable regions, one can screen narrower libraries ofantibodies and antigen-binding fragments thereof that feature targetedmutations at specific sites within hypervariable regions. This can beaccomplished, e.g., by assembling libraries of polynucleotides encodingantibodies or antigen-binding fragments thereof that encode randommutations only at particular sites within hypervariable regions. Thesepolynucleotides can then be expressed in, e.g., filamentous phage,bacterial cells, yeast cells, mammalian cells, or in vitro using, e.g.,ribosome display, mRNA display, or cDNA display techniques in order toscreen for antibodies or antigen-binding fragments thereof thatspecifically bind TNFR2 epitopes that promote receptor activation (e.g.,peptides containing the sequence of any one of SEQ ID NOs: 1-341, suchas SEQ ID NOs: 3, 11, 61, or 87, and particularly those that contain theKCSPG motif, as in SEQ ID NOs: 53, 69, 75, 118, and 233) with improvedbinding affinity. Yeast display is particularly well-suited for affinitymaturation, and has been used previously to improve the affinity of asingle chain antibody to a K_(D) of 48 fM (see Boder et al., Proc NatlAcad Sci USA 97:10701 (2000)); the disclosure of which is incorporatedherein by reference.

Additional in vitro techniques that can be used for the generation andaffinity maturation of agonistic TNFR2 antibodies of the inventioninclude the screening of combinatorial libraries of antibodies orantigen-binding fragments thereof for functional molecules capable ofspecifically binding TNFR2-derived peptides (e.g., a peptide having theamino acid sequence of any one of SEQ ID NOs: 1-341, such as SEQ ID NOs:3, 11, 61, or 87, and particularly a peptide containing the KCSPG motif,such as a peptide having the amino acid sequence of any one of SEQ IDNOs: 53, 69, 75, 118, and 233). Combinatorial antibody libraries can beobtained, e.g., by expression of polynucleotides encoding randomizedhypervariable regions of an antibody or antigen-binding fragment thereofin a eukaryotic or prokaryotic cell. This can be achieved, e.g., usinggene expression techniques described herein or known in the art.Heterogeneous mixtures of antibodies can be purified, e.g., by Protein Aor Protein G selection, sizing column chromatography), centrifugation,differential solubility, and/or by any other standard technique for thepurification of proteins. Libraries of combinatorial libraries thusobtained can be screened, e.g., by incubating a heterogeneous mixture ofthese antibodies with a peptide derived from TNFR2 that has beenimmobilized to a surface (e.g., a peptide having the amino acid sequenceof any one of SEQ ID NOs: 1-341 immobilized to the surface of asolid-phase resin or a well of a microtiter plate) for a period of timesufficient to allow antibody-antigen-binding. Non-binding antibodies orfragments thereof can be removed by washing the surface with anappropriate buffer (e.g., a solution buffered at physiological pH(approximately 7.4) and containing physiological salt concentrations andionic strength, and optionally containing a detergent, such asTWEEN-20). Antibodies that remain bound can subsequently be detected,e.g., using an ELISA-based detection protocol (see, e.g., U.S. Pat. No.4,661,445; the disclosure of which is incorporated herein by reference).

Additional techniques for screening combinatorial libraries ofantibodies for those that specifically bind TNFR2-derived peptides(e.g., a peptide containing the amino acid sequence of any one of SEQ IDNOs: 1-341, such as SEQ ID NOs: 3, 11, 61, or 87, and particularly apeptide containing the KCSPG motif, such as a peptide having the aminoacid sequence of any one of SEQ ID NOs: 53, 69, 75, 118, and 233)include the screening of one-bead-one-compound libraries of antibodyfragments. Antibody fragments can be chemically synthesized on a bead(e.g., using established split-and-pool solid phase peptide synthesisprotocols) composed of a hydrophilic, water-swellable material such thateach bead displays a single antibody fragment. Heterogeneous beadmixtures can then be incubated with a TNFR2-derived peptide that isoptionally labeled with a detectable moiety (e.g., a fluorescent dye) orthat is conjugated to an epitope tag (e.g., biotin, avidin, FLAG tag, HAtag) that can later be detected by treatment with a complementary tag(e.g., avidin, biotin, anti-FLAG antibody, anti-HA antibody,respectively). Beads containing antibody fragments that specificallybind a TNFR2-derived peptide (e.g., a peptide containing the amino acidsequence of any one of SEQ ID NOs: 1-341, such as SEQ ID NOs: 3, 11, 61,or 87, and particularly a peptide containing the KCSPG motif, such as apeptide having the amino acid sequence of any one of SEQ ID NOs: 53, 69,75, 118, and 233) can be identified by analyzing the fluorescentproperties of the beads following incubation with afluorescently-labeled antigen or complementary tag (e.g., by confocalfluorescent microscopy or by fluorescence-activated bead sorting; see,e.g., Muller et al., J. Biol. Chem., 16500-16505 (1996); the disclosureof which is incorporated herein by reference). Beads containing antibodyfragments that specifically bind TNFR2-derived peptides can thus beseparated from those that do not contain high-affinity antibodyfragments. The sequence of an antibody fragment that specifically bindsa TNFR2-derived peptide can be determined by techniques known in theart, including, e.g., Edman degradation, tandem mass spectrometry,matrix-assisted laser-desorption time-of-flight mass spectrometry(MALDI-TOF MS), nuclear magnetic resonance (NMR), and 2D gelelectrophoresis, among others (see, e.g., WO 2004/062553; the disclosureof which is incorporated herein by reference).

Negative Screens of Antibodies or Antigen-Binding Fragments

In addition to the above-described methods for screening for an antibodyor antibody fragment that specifically binds to an epitope derived fromhuman TNFR2 containing the KCSPG motif (or an equivalent of this epitopein a non-human mammal TNFR2), one can additionally perform negativescreens in order to eliminate antibodies or antibody fragments that mayalso bind an epitope that contains the KCRPG motif (e.g., a peptidecontaining residues 130-149 of SEQ ID NO: 366 (KQEGCRLCAPLRKCRPGFGV, SEQID NO: 357; or an equivalent of this epitope in a non-human mammalTNFR2).

In addition, antibodies or antibody fragments can also be screened toeliminate antibodies or antigen-binding fragments that specifically bindto a TNFR superfamily member other than TNFR2, such as TNFR1, RANK,CD30, CD40, Lymphotoxin beta receptor (LT-PR), OX40, Fas receptor, Decoyreceptor 3, CD27, 4-1 BB, Death receptor 4, Death receptor 5, Decoyreceptor 1, Decoy receptor 2, Osteoprotegrin, TWEAK receptor, TACI, BAFFreceptor, Herpesvirus entry mediator, Nerve growth factor receptor,B-cell maturation antigen, Glucocorticoid-induced TNFR-related, TROY,Death receptor 6, Death receptor 3, or Ectodysplasin A2 receptor. Thiscan be accomplished using any of the above-described methods orvariations thereof, e.g., such that the antibodies or antibody fragmentsbeing screened are those that were previously identified as beingcapable of specifically binding a peptide containing one or moreresidues of the KCSPG sequence (e.g., at least the KCS sequence).Exemplary techniques useful for a negative screen include thosedescribed above or known in the art, such as phage display, yeastdisplay, bacterial display, ribosome display, mRNA display, cDNAdisplay, or surface-based combinatorial library screens (e.g., in anELISA format). This screening technique represents a useful strategy foridentifying an agonistic TNFR2 antibody or antibody fragment of theinvention that does not bind, e.g., another TNFR superfamily member oran epitope within TNFR2 that contains the KCRPG sequence.

Immunization of a Non-Human Mammal

Another strategy that can be used to produce agonistic TNFR2 antibodiesand antigen-binding fragments thereof of the invention includesimmunizing a non-human mammal with an antigen that contains the KCSPGmotif (or an equivalent of this epitope in a non-human mammal TNFR2).Examples of non-human mammals that can be immunized in order to produceagonistic TNFR2 antibodies and fragments thereof of the inventioninclude rabbits, mice, rats, goats, guinea pigs, hamsters, horses, andsheep, as well as non-human primates. For instance, establishedprocedures for immunizing primates are known in the art (see, e.g., WO1986/6004782; the disclosure of which is incorporated herein byreference). Immunization represents a robust method of producingmonoclonal antibodies by exploiting the antigen specificity of Blymphocytes.

For example, monoclonal antibodies can be prepared by theKohler-Millstein procedure (described, e.g., in EP 0110716; thedisclosure of which is incorporated herein by reference), in whichspleen cells from a non-human animal (e.g., a primate) immunized with apeptide that presents a TNFR2-derived antigen that promotes receptoractivation (e.g., a peptide containing the amino acid sequence of anyone of SEQ ID NOs: 1-341, such as SEQ ID NOs: 3, 11, 61, or 87, andparticularly a peptide containing the KCSPG motif, such as a peptidehaving the amino acid sequence of any one of SEQ ID NOs: 53, 69, 75,118, and 233). A clonally-expanded B lymphocyte produced by immunizationcan be isolated from the serum of the animal and subsequently fused witha myeloma cell in order to form a hybridoma. Hybridomas are particularlyuseful agents for antibody production, as these immortalized cells canprovide a lasting supply of an antigen-specific antibody. Antibodiesfrom such hybridomas can subsequently be isolated using techniques knownin the art, e.g., by purifying the antibodies from the cell culturemedium by affinity chromatography, using reagents such as Protein A orProtein G.

Antibody Conjugates

Prior to administration of agonistic TNFR2 antibodies or fragmentsthereof of the invention to a mammalian subject (e.g., a human), it maybe desirable to conjugate the antibody or fragment thereof to a secondmolecule, e g., to modulate the activity of the antibody in vivo.Agonistic TNFR2 antibodies and fragments thereof can be conjugated toother molecules at either the N-terminus or C-terminus of a light orheavy chain of the antibody using any one of a variety of establishedconjugation strategies that are well-known in the art. Examples of pairsof reactive functional groups that can be used to covalently tether anagonistic TNFR2 antibody or fragment thereof to another moleculeinclude, without limitation, thiol pairs, carboxylic acids and aminogroups, ketones and amino groups, aldehydes and amino groups, thiols andα,β-unsaturated moieties (such as maleimides or dehydroalanine), thiolsand alpha-halo amides, carboxylic acids and hydrazides, aldehydes andhydrazides, and ketones and hydrazides.

Agonistic TNFR2 antibodies and fragments thereof of the invention can becovalently appended directly to another molecule by chemical conjugationas described. Alternatively, fusion proteins containing agonistic TNFR2antibodies and fragments thereof of the invention can be expressedrecombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell).This can be accomplished, for example, by incorporating a polynucleotideencoding the fusion protein into the nuclear genome of a cell (e.g.,using techniques described herein or known in the art). Optionally,antibodies and fragments thereof of the invention can be joined to asecond molecule by forming a covalent bond between the antibody and alinker. This linker can then be subsequently conjugated to anothermolecule, or the linker can be conjugated to another molecule prior toligation to the TNFR2 antibody or fragment thereof. Examples of linkersthat can be used for the formation of a conjugate include polypeptidelinkers, such as those that contain naturally occurring or non-naturallyoccurring amino acids. In certain cases, it may be desirable to includeD-amino acids in the linker, as these residues are not present innaturally-occurring proteins and are thus more resistant to degradationby endogenous proteases. Fusion proteins containing polypeptide linkerscan be made using chemical synthesis techniques, such as those describedherein, or through recombinant expression of a polynucleotide encodingthe fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell).Linkers can be prepared using a variety of strategies that are wellknown in the art, and depending on the reactive components of thelinker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysisunder acidic conditions, hydrolysis under basic conditions, oxidation,disulfide reduction, nucleophilic cleavage, or organometallic cleavage(see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012)).

Drug-Antibody Conjugates

An agonistic TNFR2 antibody or antigen-binding fragment thereof of theinvention can additionally be conjugated to, admixed with, oradministered separately from a therapeutic agent, such as a cytotoxicmolecule. Such conjugates of the invention may be applicable to, e.g.,the treatment or prevention of a disease associated with autoreactivecytotoxic T-cell activity. In these cases, antibody-drug conjugates ofthe invention may bind a TNFR2 receptor on the surface of anautoreactive T-cell and induce cell death due to the activity of theconjugated cytotoxic agent. Exemplary cytotoxic agents that can beconjugated to, admixed with, or administered separately from anagonistic TNFR2 antibody include, without limitation, antineoplasticagents such as: acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; adriamycin; aldesleukin; altretamine; ambomycin;a. metantrone acetate; aminoglutethimide; amsacrine; anastrozole;anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin;batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafidedimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine;busulfan; cactinomycin; calusterone; camptothecin; caracemide;carbetimer; carboplatin; carmustine; carubicin hydrochloride;carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin;cladribine; combretestatin a-4; crisnatol mesylate; cyclophosphamide;cytarabine; dacarbazine; daca (n-[2-(dimethyl-amino) ethyl]acridine-4-carboxamide); dactinomycin; daunorubicin hydrochloride;daunomycin; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; dolasatins; doxorubicin; doxorubicinhydrochloride; droloxifene; droloxifene citrate; dromostanolonepropionate; duazomycin; edatrexate; eflornithine hydrochloride;ellipticine; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; ethiodized oili 131; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; 5-fdump; flurocitabine; fosquidone; fostriecinsodium; gemcitabine; gemcitabine hydrochloride; gold au 198;homocamptothecin; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interferon alfa-2a; interferon alfa-2b; interferon alfa-nl;interferon alfa-n3; interferon beta-i a; interferon gamma-i b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peploycinsulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; rhizoxin; rhizoxin d; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; strontium chloride sr 89; sulofenur;talisomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; thymitaq; tiazofurin; tirapazamine;tomudex; top53; topotecan hydrochloride; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine; vinblastine sulfate; vincristine;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2′deoxyformycin; 9-aminocamptothecin; raltitrexed;N-propargyl-5,8-dideazafolic acid;2chloro-2′-arabino-fluoro-2′-deoxyadenosine; 2-chloro-2′-deoxyadenosine;anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfurmustard; nitrogen mustard (mechlor ethamine); cyclophosphamide;melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea(MNU); N, N′-Bis (2-chloroethyl)-N-nitrosourea (BCNU);N-(2-chloroethyl)-N′ cyclohexyl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl) ethylphosphonate-N-nitrosourea(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; cisplatin;carboplatin; ormaplatin; oxaliplatin; C1-973; DWA 2114R; JM216; JM335;Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-mercaptopurine; 6-thioguanine; hypoxanthine; teniposide 9-aminocamptothecin; topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans retinol;14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-hydroxyphenyl)retinamide; 13-cis retinoic acid; 3-methyl TTNEB; 9-cis retinoic acid;fludarabine (2-F-ara-AMP); or 2-chlorodeoxyadenosine (2-Cda).

Other cytotoxic agents that can be conjugated to, admixed with, oradministered separately from an agonistic TNFR2 antibody orantigen-binding fragment thereof of the invention in order to treat orprevent, e.g., the progression of a disease associated with aberrantcytotoxic T-cell proliferation include, but are not limited to,20-pi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycinB2; bropirimine; budotitane; buthionine sulfoximine; calcipotriol;calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin);canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2′deoxycoformycin(DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; discodermolide;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epothilones (A, R=H; B,R=Me); epithilones; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide; etoposide4′-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide;homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin;idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones;imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol; irinotecan; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; rnerbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; ifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mithracin;mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxinfibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; monoclonal antibody, human chorionic gonadotrophin;monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multipledrug resistance gene inhibitor; multiple tumor suppressor 1-basedtherapy; mustard anticancer agent; mycaperoxide B; mycobacterial cellwall extract; myriaporone; N-acetyldinaline; N-substituted benzamides;nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin;nartograstim; nedaplatin; nemorubicin; neridronic acid; neutralendopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxideantioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone;oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oralcytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin;paclitaxel analogues; paclitaxel derivatives; palauamine;palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin;pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium;pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol;phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetinB; plasminogen activator inhibitor; platinum complex; platinumcompounds; platinum-triamine complex; podophyllotoxin; porfimer sodium;porfiromycin; propyl bis-acridone; prostaglandin J2; proteasomeinhibitors; protein A-based immune modulator; protein kinase Cinhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethyleneconjugate; raf antagonists; raltitrexed; ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU;sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescencederived inhibitor 1; sense oligonucleotides; signal transductioninhibitors; signal transduction modulators; single chain antigen-bindingprotein; sizofiran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide;tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;telomerase inhibitors; temoporfin; temozolomide; teniposide;tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide;thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin;thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone;tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan;topsentin; toremifene; totipotent stem cell factor; translationinhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growthinhibitory factor; urokinase receptor antagonists; vapreotide; variolinB; vector system, erythrocyte gene therapy; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

Labeled TNFR2 Antibodies or Antigen-Binding Fragments

Agonistic TNFR2 antibodies or antigen-binding fragments thereof may beconjugated to another molecule, such as an epitope tag, e.g., for thepurpose of purification or detection. Examples of such molecules thatare useful in protein purification include those that present structuralepitopes capable of being recognized by a second molecule. This is acommon strategy that is employed in protein purification by affinitychromatography, in which a molecule is immobilized on a solid supportand exposed to a heterogeneous mixture containing a target proteinconjugated to a molecule capable of binding the immobilized compound.Examples of epitope tag molecules that can be conjugated to agonisticTNFR2 antibodies or fragments thereof, e.g., for the purposes ofmolecular recognition include, without limitation, maltose-bindingprotein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, amyc-tag, human influenza hemagglutinin (HA) tag, biotin, streptavidin.Conjugates containing the epitopes presented by these molecules arecapable of being recognized by such complementary molecules as maltose,glutathione, a nickel-containing complex, an anti-FLAG antibody, ananti-myc antibody, an anti-HA antibody, streptavidin, or biotin,respectively. For example, one can purify an agonistic TNFR2 antibody orfragment thereof of the invention that has been conjugated to an epitopetag from a complex mixture of other proteins and biomolecules (e.g.,DNA, RNA, carbohydrates, phospholipids, etc) by treating the mixturewith a solid phase resin containing an complementary molecule that canselectively recognize and bind the epitope tag of the TNFR2 antibody orfragment thereof. Examples of solid phase resins include agarose beads,which are compatible with purifications in aqueous solution.

A TNFR2 antibody or antigen-binding fragment thereof of the inventioncan also be covalently appended to a fluorescent molecule, e.g., todetect the antibody or antigen-binding fragment thereof by fluorimetryand/or by direct visualization using fluorescence microscopy. Exemplaryfluorescent molecules that can be conjugated to antibodies of theinvention include green fluorescent protein, cyan fluorescent protein,yellow fluorescent protein, red fluorescent protein, phycoerythrin,allophycocyanin, hoescht, 4′,6-diamidino-2-phenylindole (DAPI),propidium iodide, fluorescein, coumarin, rhodamine,tetramethylrhoadmine, and cyanine. Additional examples of fluorescentmolecules suitable for conjugation to antibodies of the invention arewell-known in the art and have been described in detail in, e.g., U.S.Pat. Nos. 7,417,131 and 7,413,874; the disclosures of each of which areincorporated by reference herein.

Agonistic TNFR2 antibodies or antigen-binding fragments thereofcontaining a fluorescent molecule are particularly useful for monitoringthe cell-surface localization properties of antibodies and fragmentsthereof of the invention. For instance, one can expose culturedmammalian cells (e.g., T-reg cells) to agonistic TNFR2 antibodies orfragments thereof of the invention that have been covalently conjugatedto a fluorescent molecule and subsequently analyze these cells usingconventional fluorescent microscopy techniques known in the art.Confocal fluorescent microscopy is a particularly powerful method fordetermining cell-surface localization of TNFR2 antibodies or fragmentsthereof, as individual planes of a cell can be analyzed in order todistinguish antibodies or fragments thereof that have been internalizedinto a cell's interior, e.g., by receptor-mediated endocytosis, fromthose that are bound to the external face of the cell membrane.Additionally, cells can be treated with TNFR2 antibodies of theinvention conjugated to a fluorescent molecule that emits visible lightof a particular wavelength (e.g., fluorescein, which fluoresces at about535 nm) and an additional fluorescent molecule that is known to localizeto a particular site on the T-reg cell surface and that fluoresces at adifferent wavelength (e.g., a molecule that localizes to CD25 and thatfluoresces at about 599 nm). The resulting emission patterns can bevisualized by confocal fluorescence microscopy and the images from thesetwo wavelengths can be merged in order to reveal information regardingthe location of the TNFR2 antibody or antigen-binding fragment thereofon the T-reg cell surface with respect to other receptors.

Bioluminescent proteins can also be incorporated into a fusion proteinfor the purposes of detection and visualization of an agonistic TNFR2antibody or fragment thereof. Bioluminescent proteins, such asLuciferase and aequorin, emit light as part of a chemical reaction witha substrate (e.g., luciferin and coelenterazine). Exemplarybioluminescent proteins suitable for use as a diagnostic sequence andmethods for their use are described in, e.g., U.S. Pat. Nos. 5,292,658;5,670,356; 6,171,809; and 7,183,092; the disclosures of each of whichare incorporated herein by reference. Agonistic TNFR2 antibodies orfragments thereof labeled with bioluminescent proteins are a useful toolfor the detection of antibodies of the invention following an in vitroassay. For instance, the presence of an agonistic TNFR2 antibody thathas been conjugated to a bioluminescent protein can be detected among acomplex mixture of additional proteins by separating the components ofthe mixture using gel electrophoresis methods known in the art (e.g.,native gel analysis) and subsequently transferring the separatedproteins to a membrane in order to perform a Western blot. Detection ofthe TNFR2 antibody among the mixture of other proteins can be achievedby treating the membrane with an appropriate Luciferase substrate andsubsequently visualizing the mixture of proteins on film usingestablished protocols.

The antibodies and fragments thereof of the invention can also beconjugated to a molecule comprising a radioactive nucleus, such that anantibody or fragment thereof of the invention can be detected byanalyzing the radioactive emission pattern of the nucleus.Alternatively, an agonistic TNFR2 antibody or fragment thereof can bemodified directly by incorporating a radioactive nucleus within theantibody during the preparation of the protein. Radioactive isotopes ofmethionine (³⁵S), nitrogen (¹⁵N), or carbon (¹³C) can be incorporatedinto antibodies or fragments thereof of the invention by, e.g.,culturing bacteria in media that has been supplemented with nutrientscontaining these isotopes. Optionally, tyrosine derivatives containing aradioactive halogen can be incorporated into an agonistic TNFR2 antibodyor fragment thereof, e.g., by culturing bacterial cells in mediasupplemented with radiolabeled tyrosine. It has been shown that tyrosinefunctionalized with a radioactive halogen at the C2 position of thephenol system are rapidly incorporated into elongating polypeptidechains using the endogenous translation enzymes in vivo (see U.S. Pat.No. 4,925,651; the disclosure of which is incorporated herein byreference). The halogens include fluorine, chlorine, bromine, iodine,and astatine. Additionally, agonistic TNFR2 antibodies or fragmentsthereof can be modified following isolation and purification from cellculture by functionalizing antibodies or fragments thereof of theinvention with a radioactive isotope. The halogens represent a class ofisotopes that can be readily incorporated into a purified protein, e.g.,by aromatic substitution at tyrosine or tryptophan via reaction of oneor more of these residues with an electrophilic halogen species.Examples of radioactive halogen isotopes include ¹⁸F, ⁷⁵Br, ⁷⁷Br, ¹²²I,¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, or ²¹¹At.

An alternative strategy for the incorporation of a radioactive isotopeis the covalent attachment of a chelating group to the agonistic TNFR2antibody or fragment thereof. Chelating groups can be covalentlyappended to an agonistic TNFR2 antibody or fragment thereof byattachment to a reactive functional group, such as a thiol, amino group,alcohol, or carboxylic acid. The chelating groups can then be modifiedto contain any of a variety of metallic radioisotopes, including,without limitation, such radioactive nuclides as ¹²⁵I, ⁶⁷Ga, ¹¹¹In,⁹⁹Tc, ¹⁶⁹Yb, ¹⁸⁶Re, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Cu,¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁹⁰Y, ⁷⁷As, ⁷²As, ⁸⁶Y, ⁸⁸Zr, ²¹¹At, ²¹²Bi, ²¹³Bi, or²²⁵Ac.

In certain cases, it may be desirable to covalently conjugate theantibodies or fragments thereof of the invention with a chelating groupcapable of binding a metal ion from heavy elements or rare earth ions,such as Gd³⁺, Fe³⁺, Mn³⁺, or Cr²⁺. Conjugates containing chelatinggroups that are coordinated to such paramagnetic metals are useful as inMRI imaging applications. Paramagnetic metals include, but are notlimited to, chromium (III), manganese (II), iron (II), iron (III),cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium(III), samarium (III), gadolinium (III), terbium (III), dysprosium(III), holmium (III), erbium (III), and ytterbium (III). In this way,agonistic TNFR2 antibodies can be detected by MRI spectroscopy. Forinstance, one can administer agonistic TNFR2 antibodies or fragmentsthereof conjugated to chelating groups bound to paramagnetic ions to amammalian subject (e.g., a human patient) in order to monitor thedistribution of the antibody following administration. This can beachieved by administration of the antibody to a patient by any of theadministration routes described herein, such as intravenously, andsubsequently analyzing the location of the administered antibody byrecording an MRI of the patient according to established protocols.

Agonistic TNFR2 antibodies or fragments thereof can additionally beconjugated to other molecules for the purpose of improving thesolubility and stability of the protein in aqueous solution. Examples ofsuch molecules include PEG, PSA, bovine serum albumin (BSA), and humanserum albumin (HSA), among others. For instance, one can conjugate anagonistic TNFR2 antibody or fragment thereof to carbohydrate moieties inorder to evade detection of the antibody or fragment thereof by theimmune system of the patient receiving treatment. This process ofhyperglycosylation reduces the immunogenicity of therapeutic proteins bysterically inhibiting the interaction of the protein with B-cellreceptors in circulation. Alternatively, agonistic TNFR2 antibodies orfragments thereof can be conjugated to molecules that prevent clearancefrom human serum and improve the pharmacokinetic profile of antibodiesof the invention. Exemplary molecules that can be conjugated to orinserted within agonistic TNFR2 antibodies or fragments thereof of theinvention so as to attenuate clearance and improve the pharmacokineticprofile of these antibodies and fragments include salvage receptorbinding epitopes. These epitopes are found within the Fc region of anIgG immunoglobulin and have been shown to bind Fc receptors and prolongantibody half life in human serum. The insertion of salvage receptorbinding epitopes into TNFR2 antibodies or fragments thereof can beachieved, e.g., as described in U.S. Pat. No. 5,739,277; the disclosureof which is incorporated herein by reference.

Modified Agonistic TNFR2 Antibodies and Antigen-Binding FragmentsThereof

In addition to conjugation to other therapeutic agents and labels foridentification or visualization, agonistic TNFR2 antibodies andfragments thereof of the invention can also be modified so as to improvetheir pharmacokinetic profile, biophysical stability, or inhibitorycapacity. For instance, any cysteine residue not involved in maintainingthe bioactive conformation of the agonistic TNFR2 antibody or fragmentthereof may be substituted with an isosteric or isolectronic amino acid(e.g., serine) in order to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cystine bond(s)may be added to the antibody or fragment thereof to improve itsstability (particularly where the antibody is an antibody fragment, suchas an Fv fragment). This can be accomplished, e.g., by altering apolynucleotide encoding the antibody heavy and light chains or apolynucleotide encoding an antibody fragment so as to encode one or moreadditional pairs of cysteine residues that can form disulfide bondsunder oxidative conditions in order to reinforce antibody tertiarystructure (see, e.g., U.S. Pat. No. 7,422,899; the disclosure of whichis incorporated herein by reference).

Another useful modification that can be made to agonistic TNFR2antibodies and fragments thereof of the invention includes altering theglycosylation profile of these antibodies and fragments thereof. Thiscan be achieved, e.g., by substituting, inserting, or deleting aminoacids in an agonistic TNFR2 antibody so as to insert or remove aglycosylation site. Glycosylation of antibodies typically occurs inN-linked or O-linked fashion. N-linked glycosylation is a processwhereby the attachment of a carbohydrate moiety to an antibody occurs atthe side chain of an asparagine residue. Consensus amino acid sequencesfor N-linked glycosylation include the tripeptide sequencesasparagine-X-serine (NXS) and asparagine-X-threonine (NXT), where X isany amino acid except proline. The insertion of either of thesetripeptide sequences in a polypeptide (e.g., an agonistic TNFR2antibody) creates a potential glycosylation site. O-linked glycosylationrefers to the attachment of one of the sugars N-acetylgalactosamine,galactose, or xylose to a hydroxyamino acid, most commonly serine orthreonine, although 5-hydroxyproline and 5-hydroxylysine are alsocompetent substrates for glycoside formation. Addition of glycosylationsites to a TNFR2 antibody can thus be accomplished by altering the aminoacid sequence of the antibody (e.g., using recombinant expressiontechniques as described herein) such that it contains one or more of theabove-described tripeptide sequences to promote N-linked glycosylation,or one or more serine or threonine residues to the sequence of theoriginal antibody engender O-linked glycosylation (see, e.g., U.S. Pat.No. 7,422,899; the disclosure of which is incorporated herein byreference).

In alternative cases, it may be desirable to modify the antibody orfragment thereof of the invention with respect to effector function,e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity(ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody.This may be achieved by introducing one or more amino acid substitutionsin an Fc region of the antibody. For instance, cysteine residues may beintroduced in the Fc region of an agonistic TNFR2 antibody or fragmentthereof (e.g., by recombinant expression techniques as describedherein), so as to facilitate additional inter-chain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave increased conformational constraint, which may foster improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described, for example, in Wolff etal., Canc. Res., 53:2560-2565 (1993); the disclosure of which isincorporated herein by reference. Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities (see Stevenson et al., Anti-Canc.Drug Des., 3:219-230 (1989); the disclosure of which is incorporatedherein by reference).

The serum half life of agonistic TNFR2 antibodies and fragments thereofof the invention can be improved in certain cases by incorporating onemore amino acid modifications, such as by altering the CH1 or CL regionof the Fab domain to introduce a salvage receptor motif, e.g., thatfound in the two loops of a CH2 domain of an Fc region of an IgG. Suchalterations are described, for instance, in U.S. Pat. Nos. 5,869,046 and6,121,022; the disclosures of which are incorporated herein byreference.

Methods of Treatment

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention are useful therapeutics for the treatment of a wide array ofimmunological disorders. Agonistic TNFR2 antibodies and fragmentsthereof can be administered to a subject, e.g., a mammalian subject,such as a human, in order to treat such conditions as autoimmunediseases, neurological diseases, metabolic diseases (e.g., diabetes),macular diseases (e.g., macular degeneration), muscular atrophy,diseases related to miscarriage, vascular diseases (e.g.,atherosclerosis), diseases related to bone loss (e.g., bone loss as aresult of menopause or osteoporosis), allergies, asthma, a blooddisorder (e.g., hemophilia), a musculoskeletal disorder, a diseaserelated to growth receptor expression or activity, obesity,graft-versus-host disease (GVHD), or an allograft rejection. AgonisticTNFR2 antibodies and antigen-binding fragments thereof of the inventioncan also be used to treat a patient in need of organ repair orregeneration, e.g., by inducing the proliferation of cells within adamaged tissue or organ. Agonistic TNFR2 antibodies and antigen-bindingfragments thereof of the invention can be administered to a mammaliansubject, such as a human, to stimulate the proliferation of T-reg cells(e.g., CD4+, CD25+, FOXP3+T-reg cells). This response can have theeffect of reducing populations of cytotoxic T-lymphocytes (e.g., CD8+T-cells) that are often associated with mounting an inappropriate immuneresponse that can cause an immunological disorder. In addition,antibodies of the invention may synergize with existing T-regproliferating agents, such as IL-2 and TNFα. For instance, antibodies orantigen-binding fragments thereof of the invention may be capable ofstimulating the proliferation of a population of T-reg cells by between1% and 100% relative to untreated cells (e.g., 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%) asdescribed in Examples 5 and 6. In certain cases, antibodies of theinvention may be capable of reducing the growth of a population of CD8+T-cells, e.g., by about 50% to about 200% relative to untreated cells(e.g., 50%, 75%, 100%, 125%, 150%, 175%, or 200%).

Agonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention can be administered to a subject, e.g., a mammalian subject,such as a human, suffering from a graft rejection. Agonistic TNFR2antibodies or antigen-binding fragments thereof of the invention maytreat graft rejections, e.g., by binding TNFR2 receptors on the surfaceof autoreactive CD8+ T-cells that bind antigens presented on the surfaceof the graft and inducing apoptosis in these CD8+ T-cells, or byinducing the expansion of T-reg cells that may subsequently eliminateautoreactive CD8+ T-cells. Examples of graft rejections that can betreated by administration of agonistic TNFR2 antibodies orantigen-binding fragments thereof of the invention include, withoutlimitation, skin graft rejection, bone graft rejection, vascular tissuegraft rejection, ligament graft rejection (e.g., cricothyroid ligamentgraft rejection, periodontal ligament graft rejection, suspensoryligament of the lens graft rejection, palmar radiocarpal ligament graftrejection, dorsal radiocarpal ligament graft rejection, ulnar collateralligament graft rejection, radial collateral ligament graft rejection,suspensory ligament of the breast graft rejection, anterior sacroiliacligament graft rejection, posterior sacroiliac ligament graft rejection,sacrotuberous ligament graft rejection, sacrospinous ligament graftrejection, inferior pubic ligament graft rejection, superior pubicligament graft rejection, anterior cruciate ligament graft rejection,lateral collateral ligament graft rejection, posterior cruciate ligamentgraft rejection, medial collateral ligament graft rejection, cranialcruciate ligament graft rejection, caudal cruciate ligament graftrejection, patellar ligament graft rejection) and organ graft rejection(e.g., heart, lung, kidney, liver, pancreas, intestine, and thymus graftrejection, among others).

Agonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention may be administered to a subject, e.g., a mammalian subject,such as a human) suffering from a graft-versus-host disease (GVHD).Exemplary graft-versus-host diseases that can be treated using thecompositions and methods of the invention include those that arises froma bone marrow transplant, as well as from the transplantation of bloodcells, such as hematopoietic stem cells, common myeloid progenitorcells, common lymphoid progenitor cells, megakaryocytes, monocytes,basophils, eosinophils, neutrophils, macrophages, T-cells, B-cells,natural killer cells, and/or dendritic cells.

Agonistic TNFR2 antibodies of the invention can be administered to asubject, e.g., a mammalian subject, such as a human, suffering from animmunological disease, e.g., in order to bind a TNFR2 receptor on thesurface of an autoreactive T-cell and induce apoptosis, and/or topromote T-reg cell growth and thus suppress the activity ofinappropriately reactive cytotoxic T-lymphocytes and B-lymphocytes inthe patient. Antibodies of the invention can be administered to asubject, e.g., via any of the routes of administration described herein.

Immunological diseases that can be treated by administration ofantibodies or antigen-binding fragments thereof of the invention includeallergies, such as food allergy, seasonal allergy, pet allergy, hives,hay fever, allergic conjunctivitis, poison ivy allergy oak allergy, moldallergy, drug allergy, dust allergy, cosmetic allergy, and chemicalallergy.

Diseases that can be treated by administration of an agonistic TNFR2antibody or antigen-binding fragment thereof of the invention includeautoimmune diseases, such as type I diabetes, alopecia areata,ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sDisease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet'sDisease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis,chronic fatigue immune dysfunction syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, Churg-Strauss Syndrome,cicatricial pemphigoid, limited scleroderma (CREST Syndrome), coldagglutinin disease, Crohn's Disease, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' Disease,Guillain-Barré Syndrome, Hashimoto's Thyroiditis, hypothyroidism,Inflammatory Bowel Disease, autoimmune lymphoproliferative syndrome(ALPS), idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus,lupus, Meniere's Disease, mixed connective tissue disease, multiplesclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia,polyarteritis nodosa, polychondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis, dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud'sPhenomenon, Reiter's Syndrome, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjögren's Syndrome, Stiff-Man syndrome,Takayasu Arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vasculitis, vitiligo, and Wegener's Granulomatosis.

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention can additionally be used to treat patients in need of organrepair or regeneration. For instance, agonistic TNFR2 antibodies orantigen-binding fragments thereof of the invention may be used tostimulate organ repair or regeneration, e.g., by binding TNFR2 on thesurface of cells within damaged tissue so as to induce TRAF2/3- and/orNFκB-mediated cell proliferation. Examples of tissues and organs thatmay be induced to regenerate by administration of an agonistic TNFR2antibody or antigen-binding fragment thereof of the invention to asubject (e.g., a mammalian subject, such as a human) include thepancreas, salivary gland, pituitary gland, kidney, heart, lung,hematopoietic system, cranial nerves, heart, blood vessels including theaorta, olfactory gland, ear, nerves, structures of the head, eye,thymus, tongue, bone, liver, small intestine, large intestine, gut,lung, brain, skin, peripheral nervous system, central nervous system,spinal cord, breast, embryonic structures, embryos, and testes.

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention can also be administered to a subject (e.g., a mammaliansubject, such as a human) in order to treat a neurological disease orcondition, such as a brain tumor, a brain metastasis, a spinal cordinjury, schizophrenia, epilepsy, Amyotrophic lateral sclerosis (ALS),Parkinson's disease, Alzheimer's disease, Huntington's disease, orstroke.

An agonistic TNFR2 antibody of the invention may also be admixed,conjugated, or administered with, or administered separately from,another agent that promotes T-reg cell proliferation. Additional agentsthat can be used to promote T-reg cell expansion include, e.g., IL-2 andTNFα, the cognate ligand for TNFR2.

Additionally or alternatively, an agonistic TNFR2 antibody orantigen-binding fragment thereof of the invention may be admixed,conjugated, or administered with, or administered separately from, animmunotherapy agent. Exemplary immunotherapy agents useful inconjunction with the compositions and methods of the invention includean anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, ananti-PD-L2 agent, a TNF-α cross-linking agent, a TRAIL cross-linkingagent, a CD27 agent, a CD30 agent, a CD40 agent, a 4-1BB agent, a GITRagent, an OX40 agent, a TRAILR1 agent, a TRAILR2 agent, a TWEAKR agent,and, e.g., agents directed toward the immunological targets described inTable 1 of Mahoney et al., Cancer Immunotherapy, 14:561-584 (2015), thedisclosure of which is incorporated herein by reference. For example,immunological target 4-1BB ligand may be targeted with an anti-4-1BBligand antibody; immunological target OX40L may be targeted with ananti-OX40L antibody; immunological target GITR may be targeted with ananti-GITR antibody; immunological target CD27 may be targeted with ananti-CD27 antibody; immunological target TL1A may be targeted with ananti-TL1A antibody; immunological target CD40L may be targeted with ananti-CD40L antibody; immunological target LIGHT may be targeted with ananti-LIGHT antibody; immunological target BTLA may be targeted with ananti-BTLA antibody; immunological target LAG3 may be targeted with ananti-LAG3 antibody; immunological target TIM3 may be targeted with ananti-TIM3 antibody; immunological target Singlecs may be targeted withan anti-Singlecs antibody; immunological target ICOS ligand may betargeted with an anti-ICOS ligand antibody; immunological target B7-H3may be targeted with an anti-B7-H3 antibody; immunological target B7-H4may be targeted with an anti-B7-H4 antibody; immunological target VISTAmay be targeted with an anti-VISTA antibody; immunological target TMIGD2may be targeted with an anti-TMIGD2 antibody; immunological target BTNL2may be targeted with an anti-BTNL2 antibody; immunological target CD48may be targeted with an anti-CD48 antibody; immunological target KIR maybe targeted with an anti-KIR antibody; immunological target LIR may betargeted with an anti-LIR antibody; immunological target ILT may betargeted with an anti-ILT antibody; immunological target NKG2D may betargeted with an anti-NKG2D antibody; immunological target NKG2A may betargeted with an anti-NKG2A antibody; immunological target MICA may betargeted with an anti-MICA antibody; immunological target MICB may betargeted with an anti-MICB antibody; immunological target CD244 may betargeted with an anti-CD244 antibody; immunological target CSF1R may betargeted with an anti-CSF1R antibody; immunological target IDO may betargeted with an anti-IDO antibody; immunological target TGFβ may betargeted with an anti-TGFβ antibody; immunological target CD39 may betargeted with an anti-CD39 antibody; immunological target CD73 may betargeted with an anti-CD73 antibody; immunological target CXCR4 may betargeted with an anti-CXCR4 antibody; immunological target CXCL12 may betargeted with an anti-CXCL12 antibody; immunological target SIRPA may betargeted with an anti-SIRPA antibody; immunological target CD47 may betargeted with an anti-CD47 antibody; immunological target VEGF may betargeted with an anti-VEGF antibody; and immunological target neuropilinmay be targeted with an anti-neuropilin antibody (see, e.g., Table 1 ofMahoney et al.).

A physician of ordinary skill in the art can readily determine aneffective amount of an agonistic TNFR2 antibody or antibody fragment foradministration to a mammalian subject (e.g., a human) in need thereof.For example, a physician could start prescribing doses of an antibody ofthe invention at levels lower than that required to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. Alternatively, a physician may begin a treatmentregimen by administering an agonistic TNFR2 antibody or antibodyfragment at a high dose and subsequently administer progressively lowerdoses until a therapeutic effect is achieved (e.g., a reduction in theproliferation of a population of CD8+ T-cells or a decrease in theperipheral secretion of IFNγ). In general, a suitable daily dose of anantibody or antigen-binding fragment thereof of the invention will be anamount of the antibody which is the lowest dose effective to produce atherapeutic effect. An antibody or antigen-binding fragment thereof ofthe invention may be administered by injection, e.g., by intravenous,intramuscular, intraperitoneal, or subcutaneous injection, optionallyproximal to the site of a target tissue. A daily dose of a therapeuticcomposition of an antibody or antigen-binding fragment thereof of theinvention may be administered as a single dose or as two, three, four,five, six or more doses administered separately at appropriate intervalsthroughout the day, week, month, or year, optionally, in unit dosageforms. While it is possible for an antibody or fragment thereof of theinvention to be administered alone, it may also be administered as apharmaceutical formulation in combination with excipients, carriers, andoptionally, additional therapeutic agents.

Antibodies or fragments thereof of the invention can be monitored fortheir ability to attenuate the progression of an immunological disease,such as an autoimmune disease, by any of a variety of methods known inthe art. For instance, a physician may monitor the response of amammalian subject (e.g., a human) to treatment with an antibody of theinvention by analyzing the quantity of IFNγ secreted by CD8+ T-cellswithin a particular patient. For example, antibodies of the inventionmay be capable of reducing IFNγ secretion by between 1% and 100% (e.g.,1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%).Alternatively, a physician may monitor the responsiveness of a subject(e.g., a human) to treatment with agonisticTNFR2 antibodies orantigen-binding fragments thereof of the invention by analyzing theT-reg cell population in the lymph of a particular subject. Forinstance, a physician may withdrawn a sample of blood from a mammaliansubject (e.g., a human) and determine the quantity or density of apopulation of T-reg cells (e.g., CD4+CD25+ FOXP3+T-reg cells orCD17+T-reg cells) using established procedures, such as fluorescenceactivated cell sorting. In these cases, high counts of T-reg cells isindicative of efficacious therapy, while lower T-reg cell counts mayindicate that the patient is to be prescribed or administered higherdosages of the TNFR2 antibody of the invention until, e.g., an idealT-reg cell count is achieved. In addition, a physician of skill in theart may monitor the effect of treatment by administration of agonisticTNFR2 antibodies of antigen-binding fragments thereof to a patientsuffering from an immunological disorder, such as an autoimmune diseasedescribed herein, by analyzing the quantity of autoreactive CD8+ T-cellswithin a lymph sample isolated from the patient. Agonistic TNFR2antibodies and antigen-binding fragments thereof of the invention mayattenuate the proliferation of autoreactive T-cells, e.g., by bindingTNFR2 at the surface of an autoreactive T-cell and inducing apoptosis,and/or by stimulating the expansion of T-reg cells that subsequentlyeliminate autoreactive T lymphocytes. Treatment with agonistic TNFR2antibodies or antigen-binding fragments thereof can lead to reducedquantities of autoreactive T-cells within the lymph isolated from apatient receiving treatment, and a rapid decline in the population ofautoreactive T-cells in a lymph sample isolated from such a patientindicates effective treatment. In cases where a lymph sample isolatedfrom a patient exhibits an autoreactive T-cell count that has notdeclined in response to agonistic TNFR2 antibody therapy, a physicianmay prescribe the patient higher doses of the antibody or anantigen-binding fragment thereof or may administer the agonistic TNFR2antibody or antigen-binding fragment thereof with higher frequency,e.g., multiple times per day, week, or month.

Pharmaceutical Compositions

Therapeutic compositions containing an agonistic TNFR2 antibody orantigen-binding fragment thereof of the invention can be prepared usingmethods known in the art. For example, such compositions can be preparedusing, e.g., physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980); incorporated herein by reference), and in a desired form,e.g., in the form of lyophilized formulations or aqueous solutions. Thecompositions can also be prepared so as to contain the active agent(e.g., an agonistic TNFR2 antibody or fragment thereof) at a desiredconcentration. For example, a pharmaceutical composition of theinvention may contain at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) active agentby weight (w/w).

Additionally, an active agent (e.g., an agonistic TNFR2 antibody orfragment thereof of the invention) that can be incorporated into apharmaceutical formulation can itself have a desired level of purity.For example, an antibody or antigen-binding fragment thereof of theinvention may be characterized by a certain degree of purity afterisolating the antibody from cell culture media or after chemicalsynthesis, e.g., of a single chain antibody fragment (e.g., scFv) byestablished solid phase peptide synthesis methods or native chemicalligation as described herein. An agonistic TNFR2 antibody of theinvention may be at least 10% pure prior to incorporating the antibodyinto a pharmaceutical composition (e.g., 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% pure).

Pharmaceutical compositions of agonistic TNFR2 antibodies of theinvention can be prepared for storage as lyophilized formulations oraqueous solutions by mixing the antibody having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients orstabilizers typically employed in the art, e.g., buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants, and other miscellaneous additives. See, e.g., Remington'sPharmaceutical Sciences, 16th edition (Osol, ed. 1980; incorporatedherein by reference). Such additives must be nontoxic to the recipientsat the dosages and concentrations employed.

Buffering Agents

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They can be present at concentration rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withTNFR2 antibodies and antigen-binding fragments thereof of the inventioninclude both organic and inorganic acids and salts thereof such ascitrate buffers {e.g., monosodium citrate-disodium citrate mixture,citric acid-trisodium citrate mixture, citric acid-monosodium citratemixture, etc.), succinate buffers {e.g., succinic acid-monosodiumsuccinate mixture, succinic acid-sodium hydroxide mixture, succinicacid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaricacid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture,tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers {e.g.,fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers {e.g., gluconic acid-sodium glyconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium glyuconatemixture, etc.), oxalate buffer {e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers {e.g., lactic acid-sodiumlactate mixture, lactic acid-sodium hydroxide mixture, lacticacid-potassium lactate mixture, etc.) and acetate buffers {e.g., aceticacid-sodium acetate mixture, acetic acid-sodium hydroxide mixture,etc.). Additionally, phosphate buffers, histidine buffers andtrimethylamine salts such as Tris can be used.

Preservatives

Preservatives can be added to a composition of the invention to retardmicrobial growth, and can be added in amounts ranging from 0.2%-1%(w/v). Suitable preservatives for use with TNFR2 antibodies andantigen-binding fragments thereof of the invention include phenol,benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzalconium halides {e.g.,chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol, and 3-pentanol. Isotonicifiers sometimes known as“stabilizers” can be added to ensure isotonicity of liquid compositionsof the invention and include polhydric sugar alcohols, for exampletrihydric or higher sugar alcohols, such as glycerin, arabitol, xylitol,sorbitol and mannitol. Stabilizers refer to a broad category ofexcipients which can range in function from a bulking agent to anadditive which solubilizes the therapeutic agent or helps to preventdenaturation or adherence to the container wall. Typical stabilizers canbe polyhydric sugar alcohols (enumerated above); amino acids such asarginine, lysine, glycine, glutamine, asparagine, histidine, alanine,ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.,organic sugars or sugar alcohols, such as lactose, trehalose, stachyose,mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glyceroland the like, including cyclitols such as inositol; polyethylene glycol;amino acid polymers; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,a-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (e.g., peptides of 10 residues or fewer); proteins such ashuman serum albumin, bovine serum albumin, gelatin or immunoglobulins;hydrophylic polymers, such as polyvinylpyrrolidone monosaccharides, suchas xylose, mannose, fructose, glucose; disaccharides such as lactose,maltose, sucrose and trisaccharides such as raffinose; andpolysaccharides such as dextran. Stabilizers can be present in the rangefrom 0.1 to 10,000 weights per part of weight active protein.

Detergents

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants can be present in a range ofabout 0.05 mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL toabout 0.2 mg/mL.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

Other Pharmaceutical Carriers

Alternative pharmaceutically acceptable carriers that can beincorporated into a composition of the invention may include dextrose,sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate,arginate, gelatin, potassium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose,methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesiumstearate, and mineral oils, but not limited to. A composition containinga TNFR2 antibody of the invention may further include a lubricant, ahumectant, a sweetener, a flavoring agent, an emulsifier, a suspendingagent, and a preservative. Details of suitable pharmaceuticallyacceptable carriers and formulations can be found in Remington'sPharmaceutical Sciences (19th ed., 1995), which is incorporated hereinby reference.

Compositions for Combination Therapy

Pharmaceutical compositions of the invention may optionally include morethan one active agent. For instance, compositions of the invention maycontain an agonistic TNFR2 antibody or fragment thereof conjugated to,admixed with, or administered separately from another pharmaceuticallyactive molecule, e.g., T-reg cell, or an additional agent that is usefulfor induction of T-reg cell expansion. For instance, an agonistic TNFR2antibody or antigen-binding fragment thereof may be admixed with one ormore additional active agents, such as IL-2 or TNFα, in order to treatan immunological disease, e.g., a disorder described herein.Alternatively, pharmaceutical compositions of the invention may beformulated for co-administration or sequential administration with oneor more additional active agents that can be used to attenuate CD8+T-cell growth. Examples of additional active agents that can be used toattenuate cytotoxic T-cell proliferation and that can be conjugated to,admixed with, or administered separately from an agonistic TNFR2antibody or antibody fragment of the invention include cytotoxic agents,e.g., those described herein.

Agonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention can also be admixed with, co-administered with, oradministered separately from Bacillus Calmette-Guérin (BCG), a bacterialstrain that has been used to treat a variety of immunological disorders,such as type I diabetes, multiple sclerosis, scleroderma, Sjogren'sdisease, systemic lupus erythematosus, Grave's disease, hypothyroidism,Crohn's disease, colititis, an autoimmune skin disease, and rheumatoidarthritis, among others. For instance, a physician of skill in the artmay prescribe a patient presenting with an immunological disorder (e.g.,one of those described above, such as type I diabetes or rheumatoidarthritis) a therapeutic regimen that includes an agonistic TNFR2antibody or antigen-binding fragment thereof of the invention incombination with BCG. The agonistic TNFR2 antibody or antigen-bindingfragment thereof may be co-administered with BCG, e.g., by an injectionroute described herein. Alternatively, the agonistic TNFR2 antibody orantigen-binding fragment thereof of the invention may be administeredseparately from a BCG-containing composition. The use of BCG to treatimmunological disorders has been described, e.g., in U.S. Pat. No.6,660,487; and in U.S. Pat. No. 6,599,710; the disclosures of each ofwhich are incorporated herein by reference.

Blood-Brain Barrier Penetration

In certain embodiments, agonistic TNFR2 antibodies or antigen-bindingfragments thereof of the invention can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compositions of the invention cross the BBB (if desired),they can be formulated, for example, in liposomes. Methods ofmanufacturing liposomes have been described, e.g., U.S. Pat. Nos.4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one ormore moieties that are selectively transported into specific cells ororgans, thereby enhancing targeted drug delivery (see, e.g., V. V.Ranade, J. Clin. Pharmacol. 29:685, 1989)). Exemplary targeting moietiesinclude, e.g., folate or biotin (see, e.g., U.S. Pat. No. 5,416,016);mannosides (Umezawa et al. (Biochem. Biophys. Res. Commun. 153:1038,1988)); antibodies (P. G. Bloeman et al. (FEBS Lett. 357:140, 1995); M.Owais et al. (Antimicrob. Agents Chemother. 39:180, 1995)); surfactantprotein A receptor (Briscoe et al. (Am. J. Physiol. 1233:134, 1995));the disclosures of each of which are incorporated herein by reference.

Routes of Administration and Dosing

Agonistic TNFR2 antibodies and antigen-binding fragments thereof of theinvention can be administered to a mammalian subject (e.g., a human) bya variety of routes such as orally, transdermally, subcutaneously,intranasally, intravenously, intramuscularly, intraocularly,intratumorally, parenterally, topically, intrathecally andintracerebroventricularly. The most suitable route for administration inany given case will depend on the particular antibody or antigen-bindingfragment administered, the patient, pharmaceutical formulation methods,administration methods (e.g., administration time and administrationroute), the patient's age, body weight, sex, severity of the diseasesbeing treated, the patient's diet, and the patient's excretion rate.

The effective dose of an agonistic TNFR2 antibody or antigen-bindingfragment thereof of the invention can range from about 0.0001 to about100 mg/kg of body weight per single (e.g., bolus) administration,multiple administrations or continuous administration, or to achieve aserum concentration of 0.0001-5000 pg/mL serum concentration per single(e.g., bolus) administration, multiple administrations or continuousadministration, or any effective range or value therein depending on thecondition being treated, the route of administration and the age,weight, and condition of the subject. In certain embodiments, e.g., forthe treatment of cancer, each dose can range from about 0.0001 mg toabout 500 mg/kg of body weight. For instance, a pharmaceuticalcomposition of the invention may be administered in a daily dose in therange of 0.001-100 mg/kg (body weight). The dose may be administered oneor more times (e.g., 2-10 times) per day, week, month, or year to amammalian subject (e.g., a human) in need thereof.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in an embodiment, a therapeutic composition ofthe invention can be administered with a needleless hypodermic injectiondevice, such as the devices disclosed in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or U.S. Pat. No.4,596,556. Examples of well-known implants and modules useful in theinvention include those described in U.S. Pat. No. 4,487,603; whichdiscloses an implantable micro-infusion pump for dispensing medicationat a controlled rate; U.S. Pat. No. 4,486,194; which discloses atherapeutic device for administering medicaments through the skin; U.S.Pat. No. 4,447,233; which discloses a medication infusion pump fordelivering medication at a precise infusion rate; U.S. Pat. No.4,447,224; which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196; whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196; which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

Kits Containing Agonistic TNFR2 Antibodies and Antigen-Binding FragmentsThereof

This invention also includes kits that contain agonistic TNFR2antibodies or antigen-binding fragments thereof. The kits providedherein may contain any of the agonistic TNFR2 antibodies and fragmentsthereof described above, as well as any of the polynucleotides encodingthese antibodies, vectors containing these polynucleotides, or cellsengineered to express and secrete antibodies of the invention (e.g.,prokaryotic or eukaryotic cells). A kit of this invention may includereagents that can be used to produce the compositions of the invention(e.g., agonistic TNFR2 antibodies, conjugates containing agonistic TNFR2antibodies, polynucleotides encoding agonistic TNFR2 antibodies, andvectors containing these polynucleotides). Optionally, kits of theinvention may include reagents that can induce the expression ofagonistic TNFR2 antibodies within cells (e.g., mammalian cells), such asdoxycycline or tetracycline. In other cases, a kit of the invention maycontain a compound capable of binding and detecting a fusion proteinthat contains an agonistic TNFR2 antibody and an epitope tag. Forinstance, in such cases a kit of the invention may contain maltose,glutathione, a nickel-containing complex, an anti-FLAG antibody, ananti-myc antibody, an anti-HA antibody, biotin, or streptavidin.

Kits of the invention may also include reagents that are capable ofdetecting an agonistic TNFR2 antibody or fragment thereof directly.Examples of such reagents include secondary antibodies that selectivelyrecognize and bind particular structural features within the Fc regionof an agonistic TNFR2 antibody of the invention. Kits of the inventionmay contain secondary antibodies that recognize the Fc region of anagonistic TNFR2 antibody and that are conjugated to a fluorescentmolecule. These antibody-fluorophore conjugates provide a tool foranalyzing the localization of agonistic TNFR2 antibodies, e.g., in aparticular tissue or cultured mammalian cell using establishedimmunofluorescence techniques. In certain cases, kits of the inventionmay include additional fluorescent compounds that exhibit knownsub-cellular localization patterns. These reagents can be used incombination with another antibody-fluorophore conjugate, e.g., one thatspecifically recognizes a different receptor on the cell surface inorder to analyze the localization of an agonistic TNFR2 antibodyrelative to other cell-surface proteins.

Kits of the invention may also contain a reagent that can be used forthe analysis of a patient's response to treatment by administration ofagonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention. For instance, kits of the invention may include an agonisticTNFR2 antibody and one or more reagents that can be used to determinethe quantity of T-reg cells in a blood sample withdrawn from a subject(e.g., a human) that is undergoing treatment with an antibody of theinvention. Such a kit may contain, e.g., antibodies that selectivelybind cell-surface antigens presented by T-reg cells, such as CD4 andCD25. Optionally, these antibodies may be labeled with a fluorescentdye, such as fluorescein or tetramethylrhodamine, in order to facilitateanalysis of a population of T-reg cells by fluorescence-activated cellsorting (FACS) methods known in the art. Kits of the invention mayoptionally contain one or more reagents that can be used to quantify apopulation of cytotoxic T-lymphocytes, e.g., in order to determine theeffectiveness of an agonistic TNFR2 antibody of the invention inattenuating CD8+ T-cell proliferation. For instance, kits of theinvention may contain an antibody that selectively binds cell-surfacemarkers on the surface of a cytotoxic T-cell, such as CD8 or CD3.Optionally, these antibodies may be labeled with fluorescent moleculesso as to enable quantitation by FACS analysis.

A kit of the invention may also contain one or more reagents useful fordetermining the affinity and selectivity of an agonistic TNFR2 antibodyor antigen-binding fragment thereof of the invention for one or morepeptides derived from TNFR2 (e.g., a peptide containing the sequence ofany one of SEQ ID NOs: 1-341, such as SEQ ID NOs: 3, 11, 61, or 87, andparticularly those that contain the KCSPG motif, as in SEQ ID NOs: 53,69, 75, 118, and 233). For instance, a kit may contain an agonisticTNFR2 antibody and one or more reagents that can be used in an ELISAassay to determine the K_(D) of an antibody of the invention for one ormore peptides that present a TNFR2 epitope in a conformation similar tothat of the epitope in the native protein. A kit may contain, e.g., amicrotiter plate containing wells that have been previously conjugatedto avidin, and may contain a library of TNFR2-derived peptides, each ofwhich conjugated to a biotin moiety. Such a kit may optionally contain asecondary antibody that specifically binds to the Fc region of anagonistic TNFR2 antibody of the invention, and the secondary antibodymay be conjugated to an enzyme (e.g., horseradish peroxidase) thatcatalyzes a chemical reaction that results in the emission ofluminescent light.

Kits of the invention may also contain agonistic TNFR2 antibodies orantigen-binding fragments thereof of the invention and reagents that canbe conjugated to such an antibody, including those previously described(e.g., a cytotoxic agent, a fluorescent molecule, a bioluminescentmolecule, a molecule containing a radioactive isotope, a moleculecontaining a chelating group bound to a paramagnetic ion, etc). Thesekits may additionally contain instructions for how the conjugation of anagonistic TNFR2 antibody of the invention to a second molecule, such asthose described above, can be achieved.

A kit of the invention may also contain a vector containing apolynucleotide that encodes an agonistic TNFR2 antibody or fragmentthereof, such as any of the vectors described herein. Alternatively, akit may include mammalian cells (e.g., CHO cells) that have beengenetically altered to express and secrete agonistic TNFR2 antibodies orfragments thereof from the nuclear genome of the cell. Such a kit mayalso contain instructions describing how expression of the agonisticTNFR2 antibody or fragment thereof from a polynucleotide can be induced,and may additionally include reagents (such as, e.g., doxycycline ortetracycline) that can be used to promote the transcription of thesepolynucleotides. Such kits may be useful for the manufacture ofagonistic TNFR2 antibodies or antigen-binding fragments thereof of theinvention.

Other kits of the invention may include tools for engineering aprokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21(DE3) E. colicell) so as to express and secrete an agonistic TNFR2 antibody orfragment thereof of the invention from the nuclear genome of the cell.For example, a kit may contain CHO cells stored in an appropriate mediaand optionally frozen according to methods known in the art. The kit mayalso provide a vector containing a polynucleotide that encodes anuclease (e.g., such as the CRISPER/Cas, zinc finger nuclease, TALEN,ARCUS™ nucleases described herein) as well as reagents for expressingthe nuclease in the cell. The kit can additionally provide tools formodifying the polynucleotide that encodes the nuclease so as to enableone to alter the DNA sequence of the nuclease in order to direct thecleavage of a specific target DNA sequence of interest. Examples of suchtools include primers for the amplification and site-directedmutagenesis of the polynucleotide encoding the nuclease of interest. Thekit may also include restriction enzymes that can be used to selectivelyexcise the nuclease-encoding polynucleotide from the vector andsubsequently re-introduce the modified polynucleotide back into thevector once the user has modified the gene. Such a kit may also includea DNA ligase that can be used to catalyze the formation of covalentphosphodiester linkages between the modified nuclease-encodingpolynucleotide and the target vector. A kit of the invention may alsoprovide a polynucleotide encoding an agonistic TNFR2 antibody orfragment thereof, as well as a package insert describing the methods onecan use to selectively cleave a particular DNA sequence in the genome ofthe cell in order to incorporate the polynucleotide encoding anagonistic TNFR2 antibody into the genome at this site. Optionally, thekit may provide a polynucleotide encoding a fusion protein that containsan agonistic TNFR2 antibody or fragment thereof and an additionalpolypeptide, such as, e.g., those described herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsclaimed herein may be performed, made, and evaluated, and are intendedto be purely exemplary of the invention and are not intended to limitthe scope of what the inventor regard as her invention.

Example 1. Mapping the Discrete Epitopes within TNFR2 that Interact withMR2-1

Libraries of linear, cyclic, and bicyclic peptides derived from humanTNFR2 were screened for distinct sequences within the protein thatexhibit high affinity for TNFR2 antibody MR2-1. In order to screenconformational epitopes within TNFR2, peptides from distinct regions ofthe primary protein sequence were conjugated to one another to formchimeric peptides. These peptides contained cysteine residues atstrategic positions within their primary sequences (see, e.g., FIG. 2A,SEQ ID NOs: 53, 69, 75, 118, and 233). This facilitated anintramolecular cross-linking strategy that was used to constrainindividual peptides to a one of a wide array of three dimensionalconformations. Unprotected thiols of cysteine residues were cross-linkedvia nucleophilic substitution reactions with divalent and trivalentelectrophiles, such as 2,6-bis(bromomethyl)pyridine and1,3,5-tris(bromomethyl)benzene, so as to form conformationallyrestricted cyclic and bicyclic peptides, respectively. In this way,peptides containing unique combinations of amino acids from disparateregions of the TNFR2 primary sequence were constrained so as tostructurally pre-organize epitopes that may resemble those presented inthe native TNFR2 tertiary structure. Libraries containing these peptideswere screened by immobilizing peptides to distinct regions of a solidsurface and treating the surface in turn with MR2-1, secondary antibodyconjugated to horseradish peroxidase (HRP), and HRP substrate(2,2′-azino-di-3-ethylbenzthiazoline sulfonate) in the presence ofhydrogen peroxide. The solid surface was washed in between treatmentwith successive reagents so as to remove excess or non-specificallybound materials. The luminescence of each region of each surface wassubsequently analyzed using a charge coupled device (CCD)—camera and animage processing system.

The “Constrained Libraries of Peptides on Surfaces” (CLIPS) platformused for this analysis begins with the conversion of the target protein,e.g., TNFR2, into a library of up to 10,000 overlapping peptideconstructs, using a combinatorial matrix design (Timmerman et al., J.Mol. Recognit., 20: 283-29 (2007)). On a solid carrier, a matrix oflinear peptides is synthesized, which are subsequently shaped intospatially defined CLIPS constructs. Constructs representing multipleparts of the discontinuous epitope in the correct conformation bind theantibody with high affinity, which is detected and quantified.Constructs presenting the incomplete epitope bind the antibody withlower affinity, whereas constructs not containing the epitope do notbind at all. Affinity information is used in iterative screens to definethe sequence and conformation of epitopes in detail.

Peptide Synthesis

To reconstruct epitopes of the target molecule a library of peptides wassynthesized. An amino functionalized polypropylene support was obtainedby grafting a proprietary hydrophilic polymer formulation via reactionwith t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) usingdicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) andsubsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA).Standard Fmoc-peptide synthesis was used to synthesize peptides on theamino-functionalized solid support by custom modified JANUS® liquidhandling stations (Perkin Elmer). CLIPS technology allows one tostructure peptides into single loops, double-loops, triple loops,sheet-like folds, helix-like folds and combinations thereof. CLIPStemplates are coupled to cysteine residues. The side-chains of multiplecysteines in the peptides are coupled to one or two CLIPS templates. Forexample, a 0.5 mM solution of the CLIPS template(2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20mM, pH 7.8)/acetonitrile (1:3(v/v)). This solution is added to asurface-bound peptide array. The CLIPS template will react withside-chains of two cysteines as present in the solid-phase boundpeptides of the peptide-arrays (455 wells plate with 3 μl wells). Thepeptide arrays are gently shaken in the solution for 30 to 60 minuteswhile completely covered in solution. Finally, the peptide arrays arewashed extensively with excess of H₂O and sonicated in disrupt-buffercontaining 1% SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70° C.for 30 minutes, followed by sonication in H₂O for another 45 minutes.

ELISA Screening

The binding of antibody to each of the synthesized peptides was testedin an ELISA format. Surface-immobilized peptide arrays were incubatedwith primary antibody solution (overnight at 4° C.). After washing, thepeptide arrays were incubated with a 1/1000 dilution of an appropriateantibody peroxidase conjugate (SBA) for one hour at 25° C. Afterwashing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazolinesulfonate (ABTS) and 2 μl/ml of 3 percent H₂O₂ were added. After onehour, the color development was measured. The color development wasquantified with a charge coupled device (CCD)—camera and an imageprocessing system. The values obtained from the CCD camera range from 0to 3000 mAU, similar to a standard 96-well plate ELISA-reader. Theresults are quantified and stored into the Peplab database. Occasionallya well contains an air-bubble resulting in a false-positive value, thecards are manually inspected and any values caused by an air-bubble arescored as 0.

Peptides that bound MR2-1 with high affinity are highlighted in FIG. 2A.These peptides therefore contain residues within TNFR2 that arestructurally configured into epitopes that are preferentially bound byMR2-1.

Example 2. Agonistic TNFR2 Antibodies Induce T-Reg Cell ProliferationMaterials and Methods

-   -   HUMAN T-REG FLOW™ Kit (BioLegend, Cat. No. 320401)        -   Cocktail Anti-human CD4 PE-Cy5/CD25 PE (BioLegend, Part No.            78930)        -   ALEXA FLUOR® 488 Anti-human FOXP3, Clone 259D (BioLegend,            Part No. 79467)        -   ALEXA FLUOR® 488 Mouse IgG1, k Isotype Ctrl (ICFC), Clone            MOPC-21 (BioLegend, Part No. 79486)        -   FOXP3 Fix/Perm Buffer (4×) (BioLegend, Cat. No. 421401)        -   FOXP3 Perm Buffer (10×) (BioLegend, Cat. No. 421402)    -   PE anti-human CD25, Clone: BC96 (BioLegend, Cat. No. 302606)    -   ALEXA FLUOR® 488 Anti-human FOXP3, Clone 259D (BioLegend, Cat.        No. 320212)    -   PBS pH 7.4 (1×) (Gibco Cat. No. 10010-023)    -   HBSS (1×) (Gibco Cat. No. 14175-095)    -   FBS (heat inactivated)    -   15 ml tubes    -   Bench top centrifuge with swing bucket rotor for 15 ml tubes        (set speed 1100 rpm or 200 g)

Agonistic TNFR2 antibodies (MR2-1 and 8E6.D1) were tested for theability to induce the proliferation of T-reg cells. Cultured T-reg cellswere treated with varying concentrations of the agonistic TNFR2antibodies in the presence and absence of stimulatory growth factors(e.g., TNFα) for set periods of time. T-reg cells were also cultured inthe presence of MR2-1 at various concentrations ranging, e.g., from0-250 μg/ml in the presence and absence of TNFα. As controls, T-regcells were also incubated with TNFα alone at concentrations ranging from0-100 ng/ml. Additionally, control T-reg cells were cultured in thepresence of IL-2 alone.

Following the incubation of T-reg cells under the conditions describedabove, the cell counts were determined using flow cytometry analysis.T-reg cells at a density of 0.2-1×10⁶ cells/100 μl were distributed intoa 15-ml conical tube and centrifuged for 5 minutes in order to pelletthe cells. The supernatant was discarded and cells were resuspended in100 μl of wash buffer (1×HBSS containing 2% FBS). 5 μl of PE anti-humanCD25 fluorophore-antibody conjugate were added to this mixture, and thecells were subsequently vortexed and incubated in the dark for 25minutes. The cells were then washed by adding 1 ml of wash buffer andsubsequently centrifuging for 5 minutes. The supernatant was thendiscarded and 1 ml of FoxP3 fixation/permeabilization buffer (1:4dilution of 4×FOXP3 Fix/Perm buffer in PBS) was added to the cells. Thecells were then vortexed and incubated in the dark for 20 minutes. Cellswere subsequently centrifuged for 5 minutes and supernatant wasdiscarded. Cells were then resuspended in 1 ml of fresh wash buffer,vortexed, and centrifuged for 5 minutes. Cells were subsequentlyresuspended in 1 ml of 1×FOXP3 Perm Buffer (1:10 dilution of 10×FOXP3Perm Buffer in PBS), vortexed, and incubated in the dark for 15 minutes.Following incubation, cells were centrifuged for 5 minutes andsupernatant was subsequently discarded. The cell pellet was thenresuspended in 100 μl of 1×FOXP3 Perm Buffer. At this point, 5 μl ofeither ALEXA FLUOR® 488 anti-human FOXP3 or ALEXA FLUOR® 488 mouse IgG1,k isotype control were added to the cells. Cells were then vortexed andincubated in the dark for 35 minutes. Following incubation, cells werewashed by adding 1 ml of fresh wash buffer to the cells, vortexing thecells and centrifuging for 5 minutes. The supernatant was then discardedand the cell pellet was resuspended in 0.2-0.5 ml of 1×HBSS free of FBS.Cell counts were then determined by flow cytometry analysis,

As seen in FIGS. 5 and 6, incubation of agonistic TNFR2 antibodies MR2-1and 8E6.D1 induced T-reg cell proliferation in a dose dependent manner.Strikingly, antibody 8E6.D1 is capable of synergizing with TNFα toenhance T-reg cell expansion (FIG. 6B).

Example 3. Generating Agonistic TNFR2 Antibodies by Phage Display

An exemplary method for in vitro protein evolution of agonistic TNFR2antibodies of the invention is phage display, a technique which is wellknown in the art. Phage display libraries can be created by making adesigned series of mutations or variations within a coding sequence forthe CDRs of an antibody or the analogous regions of an antibody-likescaffold (e.g., the BC, CD, and DE loops of ¹⁰Fn3 domains). The templateantibody-encoding sequence into which these mutations are introduced maybe, e.g., a naive human germline sequence as described herein. Thesemutations can be performed using standard mutagenesis techniquesdescribed herein or known in the art. Each mutant sequence thus encodesan antibody corresponding in overall structure to the template excepthaving one or more amino acid variations in the sequence of thetemplate. Retroviral and phage display vectors can be engineered usingstandard vector construction techniques as described herein or known inthe art. P3 phage display vectors along with compatible proteinexpression vectors, as is well known in the art, can be used to generatephage display vectors for antibody diversification as described herein.

The mutated DNA provides sequence diversity, and each transformant phagedisplays one variant of the initial template amino acid sequence encodedby the DNA, leading to a phage population (library) displaying a vastnumber of different but structurally related amino acid sequences. Dueto the well-defined structure of antibody hypervariable regions, theamino acid variations introduced in a phage display screen are expectedto alter the binding properties of the binding peptide or domain withoutsignificantly altering its structure.

In a typical screen, a phage library is contacted with and allowed tobind a TNFR2-derived peptide (e.g., a peptide having the sequence of anyone of SEQ ID NOs: 1-341, such as SEQ ID NOs: 3, 11, 61, or 87, andparticularly those that contain the KCSPG motif, as in SEQ ID NOs: 53,69, 75, 118, and 233), or a particular subcomponent thereof. Tofacilitate separation of binders and non-binders, it is convenient toimmobilize the target on a solid support. Phage bearing a TNFR2-bindingmoiety can form a complex with the target on the solid support whereasnon-binding phage remain in solution and can be washed away with excessbuffer. Bound phage can then liberated from the target by changing thebuffer to an extreme pH (pH 2 or pH 10), changing the ionic strength ofthe buffer, adding denaturants, or other known means. To isolate thebinding phage exhibiting the polypeptides of the present invention, aprotein elution is performed.

The recovered phage can then be amplified through infection of bacterialcells and the screening process can be repeated with the new pool thatis now depleted in non-binding antibodies and enriched for antibodiesthat bind the target peptide. The recovery of even a few binding phageis sufficient to amplify the phage for a subsequent iteration ofscreening. After a few rounds of selection, the gene sequences encodingthe antibodies or antigen-binding fragments thereof derived fromselected phage clones in the binding pool are determined by conventionalmethods, thus revealing the peptide sequence that imparts bindingaffinity of the phage to the target. During the panning process, thesequence diversity of the population diminishes with each round ofselection until desirable peptide-binding antibodies remain. Thesequences may converge on a small number of related antibodies orantigen-binding fragments thereof, typically 10-50 out of about 10⁹ to10¹⁰ original candidates from each library. An increase in the number ofphage recovered at each round of selection is a good indication thatconvergence of the library has occurred in a screen. After a set ofbinding polypeptides is identified, the sequence information can be usedto design other secondary phage libraries, biased for members havingadditional desired properties (see, e.g., WO 2014/152660; the disclosureof which is incorporated herein by reference).

Example 4. Producing a scFv TNFR2 Agonist

Antibody fragments of the invention include scFv fragments, whichconsist of the antibody variable regions of the light and heavy chainscombined in a single peptide chain. A TNFR2 antibody can be used as aframework for the development of an scFv antibody fragment byrecombinantly expressing a polynucleotide encoding the variable regionof a light chain of the TNFR2 antibody (e.g., 8E6.D1) operatively linkedto the variable region of a heavy chain of that antibody. This can beaccomplished using established mutagenesis protocols as described hereinor known in the art. This polynucleotide can then be expressed in a cell(e.g., a CHO cell) and the scFv fragment can subsequently be isolatedfrom the cell culture media.

Alternatively, scFv fragments derived from an agonistic TNFR2 antibodycan be produced by chemical synthetic methods (e.g., by Fmoc-basedsolid-phase peptide synthesis, as described herein). One of skill in theart can chemically synthesize a peptide chain consisting of the variableregion of a light chain of the TNFR2 antibody (e.g., 8E6.D1) operativelylinked to the variable region of a heavy chain of that antibody. Nativechemical ligation can be used as a strategy for the synthesis of longpeptides (e.g., greater than 50 amino acids). Native chemical ligationprotocols are known in the art and have been described, e.g., by Dawsonet al., Science, 266:776-779 (1994); the disclosure of which isincorporated herein by reference.

Example 5. Treatment of Type I Diabetes in a Human Patient byAdministration of Agonistic TNFR2 Antibodies

The agonistic TNFR2 antibodies of the invention (e.g., a humanizedversion of 8E6-D1 or an antigen-binding fragment thereof) can beadministered to a human patient in order to treat type I diabetes. Forinstance, a human patient suffering from type I diabetes can be treatedby administering an agonistic TNFR2 antibody of the invention by anappropriate route (e.g., intravenously) at a particular dosage (e.g.,between 0.001 and 100 mg/kg/day) over a course of days, weeks, months,or years. If desired, the agonistic TNFR2 antibody can beco-administered with, admixed with, or administered separately from,another therapeutic effective for treating type I diabetes, such as BCG.

The progression of type I diabetes that is treated with an agonisticTNFR2 antibody of the invention can be monitored by any one or more ofseveral established methods. A physician can monitor the patient bydirect observation in order to evaluate how the symptoms exhibited bythe patient have changed in response to treatment. A urine sampleisolated from the patient may be analyzed in order to determine thecontent of glucose in the sample, which can indicate the effectivenessof the TNFR2 antibody therapy. For instance, if the content of glucosein the urine sample is high, may indicate that the patient is to beadministered higher dosages of an agonistic TNFR2 antibody of theinvention until a minimal urine glucose concentration has beenmaintained.

Example 6. Treatment of Allograft Rejection in a Human Patient byAdministration of Agonistic TNFR2 Antibodies

The agonistic TNFR2 antibodies of the invention (e.g., a humanizedversion of 8E6-D1 or an antigen-binding fragment thereof) can beadministered to a human patient in order to treat allograft rejection.Administration of these antibodies induces the proliferation of apopulation of T-reg cells, which attenuates immune responses mounted byself-reactive cytotoxic T-cells that are associated with the rejectionof a tissue graft following transplantation. For instance, a humanpatient presenting with allograft rejection can be treated byadministering an agonistic TNFR2 antibody of the invention (e.g., aTNFR2 antibody that specifically binds an epitope containing one or moreresidues of the KCSPG sequence of TNFR2 (residues 56-60 of SEQ ID NO:366) by an appropriate route (e.g., intravenously) at a particulardosage (e.g., between 0.001 and 100 mg/kg/day) over a course of days,weeks, months, or years. If desired, the agonistic TNFR2 antibody can bemodified, e.g., by hyperglycosylation or by conjugation with PEG, so asto evade immune recognition and/or to improve the pharmacokineticprofile of the antibody.

The progression of the allograft rejection that is treated with anagonistic TNFR2 antibody of the invention can be monitored by any one ormore of several established methods. A physician can monitor the patientby direct observation in order to evaluate how the symptoms exhibited bythe patient have changed in response to treatment. A blood sample canalso be withdrawn from the patient in order to analyze the cell count ofone or more CD8+ T-cells in order to determine if the quantity of cellshas changed (e.g., decreased) in response to treatment with an agonisticTNFR2 antibody of the invention. A physician may also monitor thefluctuation in the volume of the allograft within the patient during thecourse of TNFR2 antibody therapy. Based on the results of theseanalyses, a physician may prescribe higher/lower dosages or more/lessfrequent dosing of the agonistic TNFR2 antibody in subsequent rounds oftreatment in order to preserve the allograft.

Example 7. Treatment of Rheumatoid Arthritis in a Human Patient byAdministration of Agonistic TNFR2 Antibodies

The agonistic TNFR2 antibodies of the invention (e.g., a humanizedversion of 8E6-D1 or an antigen-binding fragment thereof) can beadministered to a human patient in order to treat rheumatoid arthritis.For instance, a human patient suffering from rheumatoid arthritis can betreated by administering an agonistic TNFR2 antibody of the invention byan appropriate route (e.g., intravenously) at a particular dosage (e.g.,between 0.001 and 100 mg/kg/day) over a course of days, weeks, months,or years. If desired, the agonistic TNFR2 antibody can beco-administered with, admixed with, or administered separately from,another therapeutic effective for treating rheumatoid arthritis, such asBCG.

The progression of rheumatoid arthritis that is treated with anagonistic TNFR2 antibody of the invention can be monitored by any one ormore of several established methods. A physician can monitor the patientby direct observation in order to evaluate how the symptoms exhibited bythe patient have changed in response to treatment. For instance, aphysician of skill in the art may monitor the level of joint pain, jointstiffness, or muscle range exhibited by the patient in response to TNFR2antibody therapy. Additionally, a lymph sample isolated from the patientmay be analyzed in order to determine the quantity of autoreactive CD8+T-cells in the sample, e.g., by FACS analysis, which can indicate theeffectiveness of the TNFR2 antibody therapy. For instance, if the countof autoreactive CD8+ T-cells in the lymph sample is high, may indicatethat the patient is to be administered higher dosages of an agonisticTNFR2 antibody of the invention, e.g., until the autoreactive T-cellpopulation within the patient has been eliminated.

Example 8. Treatment of Multiple Sclerosis in a Human Patient byAdministration of Agonistic TNFR2 Antibodies

The agonistic TNFR2 antibodies of the invention (e.g., a humanizedversion of 8E6-D1 or an antigen-binding fragment thereof) can beadministered to a human patient in order to treat multiple sclerosis.For instance, a human patient suffering from multiple sclerosis can betreated by administering an agonistic TNFR2 antibody of the invention byan appropriate route (e.g., intravenously) at a particular dosage (e.g.,between 0.001 and 100 mg/kg/day) over a course of days, weeks, months,or years. If desired, the agonistic TNFR2 antibody can beco-administered with, admixed with, or administered separately from,another therapeutic effective for treating multiple sclerosis, such asBCG.

The progression of multiple sclerosis that is treated with an agonisticTNFR2 antibody of the invention can be monitored by any one or more ofseveral established methods. A physician can monitor the patient bydirect observation in order to evaluate how the symptoms exhibited bythe patient have changed in response to treatment. For instance, aphysician of skill in the art may monitor the patient to determine if heor she is exhibiting improved vision and/or coordination, fasterreflexes, increased motor activity, and/or improved cognitiveperformance in response to TNFR2 antibody therapy. If improvements inthese traits are not observed, a physician may prescribe the patienthigher doses or more frequent administration of the agonistic TNFRantibody or antigen-binding fragment thereof. Additionally, a lymphsample isolated from the patient may be analyzed in order to determinethe quantity of autoreactive CD8+T-cells in the sample, e.g., by FACSanalysis, which can indicate the effectiveness of the TNFR2 antibodytherapy. For instance, if the count of autoreactive CD8+ T-cells in thelymph sample that recognize myelin sheath-producing cells is high, thismay indicate that the patient is to be administered higher dosages of anagonistic TNFR2 antibody of the invention, e.g., until the autoreactiveT-cell population within the patient has been eliminated.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

1. An antibody or antigen-binding fragment thereof that specificallybinds human TNFR2, wherein said antibody or antigen-binding fragmentthereof specifically binds: (a) an epitope within human TNFR2 comprisingamino acids 56-60 of SEQ ID NO: 366; and/or (b) a polypeptide having theamino acid sequence of any one of SEQ ID NOs: 1-341, 346, and 367; andwherein said antibody or antigen-binding fragment thereof does notspecifically bind an epitope within human TNFR2 comprising amino acids142-146 of SEQ ID NO:
 366. 2-5. (canceled)
 6. The antibody orantigen-binding fragment thereof of claim 1, wherein said antibody orantigen-binding fragment thereof: (a) promotes proliferation of apopulation of T-regulatory (T-reg) cells, optionally in the presence ofTNFα; (b) promotes cell death in a population of CD8+ T-cells; (c)promotes an increase in the level of one or more mRNA molecules encodinga protein selected from the group consisting of cIAP2, TRAF2, Etk,VEGFR2, PI3K, Akt, a protein involved in the angiogenic pathway, an IKKcomplex, RIP, NIK, MAP3K, a protein involved in the NFkB pathway, NIK,JNK, AP-1, a MEK, MKK3, NEMO, IL2R, Foxp3, IL2, TNF, and lymphotoxin;(d) promotes an increase in the level of one or more proteins selectedfrom the group consisting of cIAP2, TRAF2, Etk, VEGFR2, PI3K, Akt, aprotein involved in the angiogenic pathway, an IKK complex, RIP, NIK,MAP3K, a protein involved in the NFkB pathway, NIK, JNK, AP-1, a MEK,MKK3, NEMO, IL2R, Foxp3, IL2, TNF, and lymphotoxin; (e) activates TNFR2signaling; (f) specifically binds TNFR2 with a K_(D) of no greater thanabout 10 nM; (g) specifically binds TNFR2 to form an antibody-antigencomplex with a k_(on) of at least about 10⁴ M⁻¹s⁻¹; (h) specificallybinds TNFR2 to form an antibody-antigen complex, and wherein saidcomplex dissociates with a k_(off) of no greater than about 10⁻³s⁻¹;and/or (i) does not specifically bind another tumor necrosis factorreceptor (TNFR) superfamily member. 7-9. (canceled)
 10. The antibody orantigen-binding fragment thereof of claim 1, wherein said antibody orantigen-binding fragment thereof specifically binds: (a) an epitopewithin human TNFR2 comprising at least five discontinuous or continuousresidues within amino acids 96-154 of SEQ ID NO: 366 (b) an epitopewithin amino acids 111-150 of SEQ ID NO: 366; (c) an epitope withinamino acids 115-142 of SEQ ID NO: 366; (d) an epitope within amino acids122-136 of SEQ ID NO: 366; (e) an epitope within amino acids 101-107 ofSEQ ID NO: 366; (f) an epitope within amino acids 48-67 of SEQ ID NO:366; and/or (g) said epitope comprising amino acids 56-60 of SEQ ID NO:366 with a K_(D) of less than about 10 nM. 11-26. (canceled)
 27. Amethod of identifying a TNFR2 agonist antibody or antigen-bindingfragment thereof comprising: (a) contacting a mixture of antibodies orfragments thereof with at least one peptide having the amino acidsequence of any one of SEQ ID NOs: 1-341, 346, and 367; and (b)separating antibodies or fragments thereof that specifically bind saidpeptide from said mixture, thereby producing an enriched antibodymixture comprising at least one said TNFR2 agonist antibody orantigen-binding fragment thereof.
 28. The method of claim 27, wherein:(a) said method comprises determining the amino acid sequence of one ormore of the antibodies or antigen-binding fragments thereof in saidenriched antibody mixture; (b) said peptide is bound to a surface; (c)said antibody or antigen-binding fragment thereof is expressed on thesurface of a phage, bacterial cell, or yeast cell or said antibody orantigen-binding fragment thereof is expressed on the surface of a phage,bacterial cell, or yeast cell; (d) said peptide is conjugated to adetectable label; (e) steps (a) and (b) are sequentially repeated one ormore times; and/or (f) the method further comprises: i) exposing saidenriched antibody mixture to at least one peptide comprising the aminoacid sequence of a TNFR superfamily member other than TNFR2, andretaining antibodies or fragments thereof that do not specifically bindsaid peptide, thereby producing a TNFR2-specific antibody mixturecomprising at least one TNFR2 agonist antibody or antigen-bindingfragment thereof that does not specifically bind a TNFR superfamilymember other than TNFR2; and/or ii) exposing said enriched antibodymixture to at least one peptide comprising amino acids 142-146 of SEQ IDNO: 366, and retaining antibodies or fragments thereof that do notspecifically bind said peptide, thereby producing an antibody mixturecomprising at least one TNFR2 agonist antibody or antigen-bindingfragment thereof that does not specifically bind a peptide comprisingamino acids 142-146 of SEQ ID NO:
 366. 29-38. (canceled)
 39. A method ofproducing a TNFR2 agonist antibody or antigen-binding fragment thereofcomprising immunizing a non-human mammal with a peptide comprising thesequence of any one of SEQ ID NOs: 1-341, 346, and 367 and collectingserum comprising said TNFR2 agonist antibody or antigen-binding fragmentthereof, wherein said antibody or antigen-binding fragment thereof iscapable of specifically binding an epitope comprising amino acids 56-60of SEQ ID NO:
 366. 40. The method of claim 39, wherein: (a) saidnon-human mammal is selected from the group consisting of a rabbit,mouse, rat, goat, guinea pig, hamster, horse, and sheep; and/or (b) saidpeptide comprises the amino acid sequence of SEQ ID NO:
 11. 41.(canceled)
 42. An antibody or antigen-binding fragment thereof that isproduced by the method of claim
 39. 43-53. (canceled)
 54. The antibodyor antigen-binding fragment thereof of claim 1, wherein said antibody orantigen-binding fragment thereof is a monoclonal antibody orantigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a human antibody or antigen-bindingfragment thereof, a humanized antibody or antigen-binding fragmentthereof, a primatized antibody or antigen-binding fragment thereof, abispecific antibody or antigen-binding fragment thereof, amulti-specific antibody or antigen-binding fragment thereof, adual-variable immunoglobulin domain, a monovalent antibody orantigen-binding fragment thereof, a chimeric antibody or antigen-bindingfragment thereof, a single-chain Fv molecule (scFv), a diabody, atriabody, a nanobody, an antibody-like protein scaffold, a domainantibody, a Fv fragment, a Fab fragment, a F(ab′)₂ molecule, or a tandemscFv (taFv).
 55. (canceled)
 56. The antibody of claim 1, wherein saidantibody comprises an immunoglobulin subtype selected from the groupconsisting of IgG, IgM, IgA, IgD, and IgE.
 57. A pharmaceuticalcomposition comprising the antibody or antigen-binding fragment thereofof claim 1 and a pharmaceutically acceptable carrier or excipient. 58.The pharmaceutical composition of claim 57, wherein said pharmaceuticalcomposition further comprises an additional therapeutic agent,optionally wherein said additional therapeutic agent is selected fromthe group consisting of TNFα and BCG. 59-61. (canceled)
 62. Apolynucleotide encoding the antibody or antigen-binding fragment thereofof claim
 1. 63. A vector comprising the polynucleotide of claim 62.64-70. (canceled)
 71. An isolated host cell comprising the vector ofclaim
 63. 72. The host cell of claim 71, wherein said host cell is: (a)a prokaryotic cell; or (b) a eukaryotic cell; optionally wherein saideukaryotic cell is a CHO cell, a DHFR CHO cell, a NSO myeloma cell, aCOS cell, a 293 cell, or a SP2/0 cell. 73-75. (canceled)
 76. A method ofproducing the antibody or antigen-binding fragment thereof of claim 1,said method comprising expressing a polynucleotide encoding saidantibody or antigen-binding fragment thereof in a host cell andrecovering the antibody or antigen-binding fragment thereof from hostcell medium.
 77. A method of inhibiting an immune response mediated by aB cell or CD8+ T cell in a subject, said method comprising administeringto the subject the antibody or antigen-binding fragment thereof ofclaim
 1. 78. A method of treating an immunological disease in a subject,said method comprising administering to the subject the antibody orantigen-binding fragment thereof of claim
 1. 79. The method of claim 78,wherein said subject is in need of a tissue or organ regeneration. 80.The method of claim 79, wherein said tissue or organ is selected fromthe group consisting of a pancreas, salivary gland, pituitary gland,kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta,olfactory gland, ear, nerves, structures of the head, eye, thymus,tongue, bone, liver, small intestine, large intestine, gut, lung, brain,skin, peripheral nervous system, central nervous system, spinal cord,breast, embryonic structures, embryos, and testes.
 81. The method ofclaim 78, wherein said immunological disease is selected from the groupconsisting of an autoimmune disease, a neurological condition, anallergy, asthma, macular degeneration, muscular atrophy, a diseaserelated to miscarriage, atherosclerosis, bone loss, a musculoskeletaldisease, obesity, a graft-versus-host disease, and an allograftrejection.
 82. The method of claim 81, wherein: (a) said autoimmunedisease is selected from the group consisting of type I diabetes,Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Syndrome,Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia, AutoimmuneHepatitis, Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, CeliacSprue-Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS),Chronic Inflammatory Demyelinating Polyneuropathy, Churg-StraussSyndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold AgglutininDisease, Crohn's Disease, Essential Mixed Cryoglobulinemia,Fibromyalgia-Fibromyositis, Graves' Disease, Guillain-Barré, Hashimoto'sThyroiditis, Hypothyroidism, Idiopathic Pulmonary Fibrosis, IdiopathicThrombocytopenia Purpura (ITP), IgA Nephropathy, Juvenile Arthritis,Lichen Planus, Lupus, Meniere's Disease, Mixed Connective TissueDisease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris,Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, PolyglandularSyndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis,Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis,Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, RheumatoidArthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome, Stiff-ManSyndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis,Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, and Wegener'sGranulomatosis; (b) said neurological condition is selected from thegroup consisting of a brain tumor, a brain metastasis, a spinal cordinjury, schizophrenia, epilepsy, Amyotrophic lateral sclerosis (ALS),Parkinson's disease, Alzheimer's disease, Huntington's disease, andstroke; (c) said allergy is selected from the group consisting of foodallergy, seasonal allergy, pet allergy, hives, hay fever, allergicconjunctivitis, poison ivy allergy oak allergy, mold allergy, drugallergy, dust allergy, cosmetic allergy, and chemical allergy; (d) saidgraft-versus-host disease arises from a bone marrow transplant or one ormore blood cells selected from the group consisting of hematopoieticstem cells, common myeloid progenitor cells, common lymphoid progenitorcells, megakaryocytes, monocytes, basophils, eosinophils, neutrophils,macrophages, T-cells, B-cells, natural killer cells, and dendriticcells; and/or (e) said allograft rejection is selected from the groupconsisting of skin graft rejection, bone graft rejection, vasculartissue graft rejection, ligament graft rejection, and organ graftrejection, optionally wherein: (i) said ligament graft rejection isselected from the group consisting of cricothyroid ligament graftrejection, periodontal ligament graft rejection, suspensory ligament ofthe lens graft rejection, palmar radiocarpal ligament graft rejection,dorsal radiocarpal ligament graft rejection, ulnar collateral ligamentgraft rejection, radial collateral ligament graft rejection, suspensoryligament of the breast graft rejection, anterior sacroiliac ligamentgraft rejection, posterior sacroiliac ligament graft rejection,sacrotuberous ligament graft rejection, sacrospinous ligament graftrejection, inferior pubic ligament graft rejection, superior pubicligament graft rejection, anterior cruciate ligament graft rejection,lateral collateral ligament graft rejection, posterior cruciate ligamentgraft rejection, medial collateral ligament graft rejection, cranialcruciate ligament graft rejection, caudal cruciate ligament graftrejection, and patellar ligament graft rejection; and/or (ii) said organgraft rejection is selected from the group consisting of heart graftrejection, lung graft rejection, kidney graft rejection, liver graftrejection, pancreas graft rejection, intestine graft rejection, andthymus graft rejection. 83-88. (canceled)
 89. The method of claim 78,wherein said method further comprises administering to said subject anadditional therapeutic agent.
 90. The method of claim 89, wherein saidadditional therapeutic agent is selected from the group consisting ofTNFα and BCG. 91-92. (canceled)
 93. The method of claim 78, wherein saidsubject is a mammal.
 94. The method of claim 93, wherein said mammal isa human.
 95. The method of claim 78, wherein said antibody is 8E6.D1 ora humanized antibody or antigen-binding fragment thereof comprising oneor more heavy chain or light chain CDRs of 8E6.D1.
 96. (canceled)
 97. Akit comprising the antibody or antigen-binding fragment thereof of claim1, a polynucleotide encoding the antibody or antigen-binding fragmentthereof, a vector comprising the polynucleotide, or a host cellcomprising the vector or polynucleotide, wherein the kit optionallycomprises a package insert that instructs a user of said kit toadminister said antibody or antigen-binding fragment thereof,polynucleotide, vector, or host cell to a human patient suffering froman immunological disease. 98-129. (canceled)
 130. The antibody orantigen-binding fragment thereof of claim 1, wherein said antibody orantigen-binding fragment thereof comprises a non-native constant region.131. The antibody or antigen-binding fragment thereof of claim 1,wherein said antibody or antigen-binding fragment thereof is anisolated, non-murine antibody. 132-133. (canceled)